U.S. patent application number 12/130299 was filed with the patent office on 2009-03-26 for methods for selection of melanocortin receptor-specific agents for treatment of obesity.
This patent application is currently assigned to Palatin Technologies, Inc.. Invention is credited to Kevin D. Burris, Shubh D. Sharma.
Application Number | 20090081197 12/130299 |
Document ID | / |
Family ID | 40471888 |
Filed Date | 2009-03-26 |
United States Patent
Application |
20090081197 |
Kind Code |
A1 |
Burris; Kevin D. ; et
al. |
March 26, 2009 |
Methods for Selection of Melanocortin Receptor-Specific Agents for
Treatment of Obesity
Abstract
Provided are methods for selecting melanocortin
receptor-specific compounds for attenuating food intake and for
treatment of specific disease conditions, including treatment of
obesity and related energy homeostasis or feeding disorders
characterized by excess weight gain, without inducing a sexual
response, and methods for selecting compounds for treatment of
sexual dysfunction. Further provided are pharmaceutical
preparations defined by such methods, and methods and preparations
for attenuating food intake and treatment of said conditions.
Inventors: |
Burris; Kevin D.;
(Indianapolis, IN) ; Sharma; Shubh D.; (Cranbury,
NJ) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
Palatin Technologies, Inc.
Cranbury
NJ
|
Family ID: |
40471888 |
Appl. No.: |
12/130299 |
Filed: |
May 30, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60924836 |
Jun 1, 2007 |
|
|
|
Current U.S.
Class: |
424/130.1 ;
435/6.11; 435/6.14; 435/7.2 |
Current CPC
Class: |
A61P 3/00 20180101; G01N
2800/02 20130101; G01N 33/566 20130101; G01N 2333/726 20130101 |
Class at
Publication: |
424/130.1 ;
435/7.2; 435/6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/53 20060101 G01N033/53; A61P 3/00 20060101
A61P003/00; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A method of selecting a compound as a candidate for attenuating
food intake which compound at least substantially does not induce
or initiate a sexual response, the method comprising the steps of:
providing a low melanocortin 4 receptor (MC4-R) density system;
providing a high density MC4-R system; determining the functional
activity of the compound in the low density MC4-R system;
determining the functional activity of the compound in the high
density MC4-R system; and selecting the compound as a candidate if
the compound is functionally inactive in the low MCR-4 density
system and is functionally active in the high density MC4-R
system.
2. The method of claim 1, wherein determining the functional
activity of the compound in the low density and high density MC4-R
system comprises determining activation of melanocortin 4 receptors
in each of the low density and high density MC4-R systems.
3. The method of claim 1, wherein determining the functional
activity of the compound in the low density and high density MC4-R
system comprises an adenylyl cyclase activity assay.
4. The method of claim 1, wherein the low density MC4-R system is a
low human MC4-R (hMC4-R) density system and the high density MC4-R
system is a high hMC4-R density system.
5. The method of claim 1, wherein the low density MC4-R system has
a melanocortin receptor density determined by receptor binding
saturation that is at least ten times lower than the melanocortin
receptor density in the high density MC4-R system.
6. The method of claim 5, wherein the low density MC4-R system has
a melanocortin receptor density determined by receptor binding
saturation that is at least fifty times lower than the melanocortin
receptor density in the high density MC4-R system.
7. The method of claim 5, wherein the low density MC4-R system has
a melanocortin receptor density determined by receptor binding
saturation that is at least one hundred times lower than the
melanocortin receptor density in the high density MC4-R system.
8. The method of claim 1, wherein both the low density MC4-R system
and the high density MC4-R system comprise mammalian cells
transformed or transfected with a gene encoding MC4-R, which gene
is expressible in the cells.
9. The method of claim 8, wherein the gene encoding for MC4-R
comprises a gene encoding for hMC4-R.
10. The method of claim 8, wherein the gene encoding for MC4-R is
under the control of a gene expression variable regulatory
system.
11. The method of claim 10, wherein the gene expression variable
regulatory system comprises a tetracycline-regulated mammalian
expression system.
12. The method of claim 5, wherein the low density MC4-R system has
a receptor density expressed as a B.sub.max receptor binding
saturation value using NDP-.alpha.-MSH of between approximately 80
and 20 fmol/mg.
13. The method of claim 5, wherein the high density MC4-R system
has a receptor density expressed as a B.sub.max receptor binding
saturation value using NDP-.alpha.-MSH of at least about 4,000
fmol/mg.
14. The method of claim 8, wherein the mammalian cells comprise
HEK-293 cells.
15. The method of claim 1, wherein the compound is functionally
inactive in the low MCR-4 density system where the compound has no
measurable intrinsic activity.
16. The method of claim 1, wherein the compound is functionally
inactive in the low MCR-4 density system and has an intrinsic
activity of less than about 0.1 (10%), based on maximal stimulation
of adenylyl cyclase achievable by the compound in the low hMC4-R
density system wherein the maximal stimulation achieved by
.alpha.-MSH or NDP-.alpha.-MSH is designated as an intrinsic
activity of 1.0 (100%).
17. The method of claim 1, wherein the compound is functionally
active in the high density MC4-R system and has an intrinsic
activity of more than about 0.1 (10%), based on maximal stimulation
of adenylyl cyclase achievable by the compound in the high hMC4-R
density system wherein the maximal stimulation achieved by
.alpha.-MSH or NDP-.alpha.-MSH is designated as an intrinsic
activity of 1.0 (100%).
18. The method of claim 17, wherein the compound is functionally
active in the high density MC4-R system and has an intrinsic
activity of more than about 0.2 (20%), based on maximal stimulation
of adenylyl cyclase achievable by the compound in the high hMC4-R
density system wherein the maximal stimulation achieved by
.alpha.-MSH or NDP-.alpha.-MSH is designated as an intrinsic
activity of 1.0 (100%).
19. The method of claim 17, wherein the compound is functionally
active in the high density MC4-R system and has an intrinsic
activity of more than about 0.3 (30%), based on maximal stimulation
of adenylyl cyclase achievable by the compound in the high hMC4-R
density system wherein the maximal stimulation achieved by
.alpha.-MSH or NDP-.alpha.-MSH is designated as an intrinsic
activity of 1.0 (100%).
20. The method of claim 17, wherein the compound is functionally
active in the high density MC4-R system and has an intrinsic
activity of more than about 0.5 (50%), based on maximal stimulation
of adenylyl cyclase achievable by the compound in the high hMC4-R
density system wherein the maximal stimulation achieved by
.alpha.-MSH or NDP-.alpha.-MSH is designated as an intrinsic
activity of 1.0 (100%).
21. The method of claim 17, wherein the compound is functionally
active in the high density MC4-R system and has an intrinsic
activity of more than about 0.7 (70%), based on maximal stimulation
of adenylyl cyclase achievable by the compound in the high hMC4-R
density system wherein the maximal stimulation achieved by
.alpha.-MSH or NDP-.alpha.-MSH is designated as an intrinsic
activity of 1.0 (100%).
22. A method of selecting a compound as a candidate for treating
sexual dysfunction, the method comprising the steps of: providing a
low density MC4-R system; providing a high density MC4-R system;
determining the functional activity of the compound in the low
density MC4-R system; determining the functional activity of the
compound in the high density MC4-R system; and selecting the
compound as a candidate if the compound is functionally active in
both the low MCR-4 density system and high density MC4-R
system.
23. The method of claim 22, wherein determining the functional
activity of the compound in the low density and high density MC4-R
system comprises determining activation of melanocortin 4 receptors
in each of the low density and high density MC4-R systems.
24. The method of claim 22, wherein determining the functional
activity of the compound in the low density and high density MC4-R
system comprises an adenylyl cyclase activity assay.
25. The method of claim 22, further comprising the step of:
determining the functional status of the compound in a transformed
MC4-R system not under the control of a gene expression variable
regulatory system, and wherein selecting the compound as a
candidate for treating sexual dysfunction further requires that the
compound is an agonist in the transformed MC4-R system not under
the control of a gene expression variable regulatory system.
26. The method of claim 25, wherein the compound is an agonist in
the transformed MC4-R system and has an intrinsic activity of more
than about 0.7 (70%), based on maximal stimulation of adenylyl
cyclase achievable by the compound in the transformed hMC4-R system
wherein the maximal stimulation achieved by .alpha.-MSH or
NDP-.alpha.-MSH is designated as an intrinsic activity of 1.0
(100%).
27. The method of claim 22, wherein the low density MC4-R system is
a low human MC4-R (hMC4-R) density system and the high density
MC4-R system is a high hMC4-R density system.
28. The method of claim 22, wherein the low density MC4-R system
has a melanocortin receptor density determined by receptor binding
saturation that is at least ten times lower than the melanocortin
receptor density in the high density MC4-R system.
29. The method of claim 28 wherein the low density MC4-R system has
a melanocortin receptor density determined by receptor binding
saturation that is at least fifty times lower than the melanocortin
receptor density in the high density MC4-R system.
30. The method of claim 28, wherein the low density MC4-R system
has a melanocortin receptor density determined by receptor binding
saturation that is at least one hundred times lower that the
melanocortin receptor density in the high density MC4-R system.
31. The method of claim 22, wherein both the low density MC4-R
system and the high density MC4-R system comprise mammalian cells
transformed or transfected with a gene encoding MC4-R, which gene
is expressible in the cells.
32. The method of claim 31, wherein the gene encoding for MC4-R
comprises a gene encoding for hMC4-R.
33. The method of claim 31, wherein the gene encoding for MC4-R is
under the control of a gene expression variable regulatory
system.
34. The method of claim 33, wherein the gene expression variable
regulatory system comprises a tetracycline-regulated mammalian
expression system that is regulatable by tetracycline or a related
regulator.
35. A method of selecting a melanotropin receptor binding compound
as a candidate for attenuating food intake which compound at least
substantially does not induce or initiate a sexual response, the
method comprising the steps of: providing a low density MC4-R
system; providing a high density MC4-R system; determining the
functional activity of the compound in the low density MC4-R
system; determining the functional activity of the compound in the
high density MC4-R system; and selecting the compound as a
candidate if the compound is functionally inactive in both the low
MCR-4 density system and the high density MC4-R system.
36. The method of claim 35, further comprising the step of:
providing a competition inhibition assay for competitive binding
with a MC4-R agonist as to MC4-R, wherein selecting the compound as
a candidate for attenuating food intake further requires that the
compound inhibits at least about 90% of the binding of the MC4-R
agonist to MC4-R.
37. The method of claim 36, where the MC4-R agonist is
.alpha.-MSH.
38. The method of claim 37, further comprising the step of:
providing an assay for determining the Ki (nM) of the compound as
to .alpha.-MSH binding to MC4-R, wherein selecting the compound as
a candidate for attenuating food intake further requires that the
compound has a Ki (nM) of less than about 100.
39. The method of claim 38, wherein the compound has a Ki (nM) of
less than about 10.
40. The method of claim 38, wherein the compound has a Ki (nM) of
less than about 1.
41. The method of claim 35, wherein the low density MC4-R system is
a low human MC4-R (hMC4-R) density system and the high density
MC4-R system is a high hMC4-R density system.
42. The method of claim 35, wherein the compound is functionally
inactive in both the low MCR-4 density system and the high density
MC4-R system wherein the compound has an intrinsic activity of less
than about 0.1 (10%), based on maximal stimulation of adenylyl
cyclase achievable by the compound in each of the low and high
MC4-R density system wherein the maximal stimulation achieved by
.alpha.-MSH or NDP-.alpha.-MSH is designated as an intrinsic
activity of 1.0 (100%).
43. A pharmaceutical composition for attenuating food intake
without substantially inducing a sexual response, comprising: an
effective amount of a melanocortin-4 receptor (MC4-R) specific
compound which is functionally inactive at a concentration of about
1000 nM or less in an adenylyl cyclase activity assay at a low
density MC4-R and which is functionally active at a concentration
of about 1000 nM or less in an adenylyl cyclase activity assay at a
high density MC4-R; and one or more of a carrier, an excipient and
an adjunct ingredient.
44. A pharmaceutical composition for attenuating food intake
without substantially inducing a sexual response, comprising: an
effective amount of a melanocortin-4 receptor (MC4-R) specific
compound which is functionally inactive at a concentration of about
1000 nM or less in an adenylyl cyclase expression assay at a low
density MC4-R and at a high density MC4-R; and one or more of a
carrier, an excipients and an adjunct ingredient.
45. A pharmaceutical composition for treatment of sexual
dysfunction, comprising: an effective amount of a melanocortin-4
receptor (MC4-R) specific compound which is functionally active at
a concentration of about 1000 nM or less in an adenylyl cyclase
activity assay at both a low density MC4-R and at a high density
MC4-R; and one or more of a carrier, an excipient and an adjunct
ingredient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/924,836 filed Jun. 1, 2007, which is herein
incorporated by reference in its entirety.
[0002] A related U.S. application entitled Compounds and Methods
for Treatment of Obesity is being filed concurrently herewith,
Attorney Docket No. 056291-5365, and the specification and claims
thereof are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention (Technical Field)
[0004] The present invention relates to screening and selection
methods for selecting melanocortin receptor-specific compounds for
attenuating food intake, such as for the treatment of obesity and
related energy homeostasis or feeding disorders characterized by
excess weight gain, which compounds do not induce a sexual
response, including a penile response, and to particular compounds
thus selected. The invention also relates to related screening and
selection methods for selecting melanocortin receptor-specific
compounds for treating sexual dysfunction, such as male erectile
dysfunction.
[0005] 2. Description of Related Art
[0006] Note that the following discussion refers to a number of
publications by author(s) and year of publication, and that due to
recent publication dates certain publications are not to be
considered as prior art vis-a-vis the present invention. Discussion
of such publications herein is given for more complete background
and is not to be construed as an admission that such publications
are prior art for patentability determination purposes.
[0007] A family of melanocortin receptor types and subtypes have
been identified, including melanocortin 1 receptors (MC1-R)
expressed on normal human melanocytes and melanoma cells,
melanocortin 2 receptors (MC2-R) for adrenocorticotropin (ACTH)
expressed in cells of the adrenal gland, melanocortin 3 and
melanocortin 4 receptors (MC3-R and MC4-R), expressed primarily in
cells in the hypothalamus, mid-brain and brainstem, and
melanocortin 5 receptors (MC5-R), expressed in a wide distribution
of tissues.
[0008] It has been hypothesized that compounds specific for MC3-R
or MC4-R are useful in regulation of energy homeostasis, including
use as agents for attenuating food intake and body weight gain, for
use in treatment of anorexia and cachexia, for treatment of
obesity, and treatment of other food intake and metabolism-related
indications. However, the mechanism of action of compounds specific
for MC3-R or MC4-R as agents for regulation of energy homeostasis
has not been fully elucidated.
[0009] Most MC4-R agonist compounds that have been evaluated for
use in attenuating food intake and body weight gain also have other
systemic effects relating to the melanocortin system, including
inducing or facilitating penile erection in males. These effects
render most MC4-R agonists unsuitable for use in treatment of
obesity. There is a need for melanocortin-specific agents which are
efficacious for treatment of obesity, but without other systemic
effects, such as induction of penile erections in males, or a
sexual response generally in mammals.
[0010] It has been shown that melanocortin receptors, including
MC4-R, can express a basal or constitutive level (i.e.,
unstimulated) of adenylyl cyclase activity, even in the absence of
a specific agonist. Adenylyl cyclase is an enzyme of the lyase
class that catalyzes the formation of 3',5'-cyclic adenosine
monophosphate (cAMP) from ATP (adenosine triphosphate, the 5'
triphosphate of adenosine). It is clear that earlier models of
receptor theory are not sufficient to explain observed results. Two
papers by T. Kenakin, among others, provide receptor models,
including a ternary complex model, specifically for G-protein
coupled receptors, which recognize that efficacy of a compound may
vary based on receptor density, efficiency of receptor coupling,
and other environmental factors relating to the receptor. (Kenakin
T. Efficacy at G-Protein-Coupled Receptors (review). Nature Reviews
1:103-110 (2002); Kenakin T. Principles: Receptor theory in
pharmacology (review). TRENDS in Pharmacol. Sci. 25:186-192
(2004)).
[0011] Most, if not all, MC4-R agonist compounds explored for
attenuating food intake also induce a sexual response, including a
penile erection response in males, and in addition all or virtually
all MC4-R agonist compounds reported for attenuating food intake or
body weight gain in animal models have shown a "rebound" effect,
with animals gaining weight equal to or, in many instances
exceeding, controls upon cessation of administration of the
compounds. There is thus a need for compounds which attenuate food
intake or body weight gain without causing a rebound effect upon
cessation of administration of the compound.
[0012] U.S. Pat. No. 5,908,609, by Lee, Huszar and Gu and assigned
to Millennium Pharmaceuticals, Inc., discloses a method for
identifying compounds that regulate body weight, including
contacting a test compound with a cell which expresses a functional
MC4-R, and determining whether the compound activates the receptor.
It is claimed that compounds that "activate" MC4-R are identified
as compounds for inducing weight loss. However, "activation" is
defined in terms of simple induction of cAMP expression in a fixed
melanocortin receptor density system, and thus this patent simply
claims a method of selecting a MC4-R agonist. Similarly, U.S. Pat.
No. 6,716,810, by Brennan and Hochgeschwender and assigned to
Eleanor Roosevelt Institute and Oklahoma Medical Research
Foundation, discloses methods of regulating body weight, such as by
administration of .alpha.-MSH, an .alpha.-MSH analog, or an
.alpha.-MSH homolog having agonist activity. Neither patent, nor
any other methods disclosed elsewhere, address the selection of a
melanocortin receptor-specific compound that is effective for
modulating feeding behavior or energy homeostasis, but which does
not induce other systemic effects, for example, which does not
induce a sexual response.
SUMMARY OF THE INVENTION
[0013] In one aspect, the invention provides a method of selecting
a compound as a candidate for attenuating food intake, such as for
treating obesity and related energy homeostasis or feeding
disorders characterized by excess weight gain, which compound at
least substantially does not induce or initiate a sexual response,
the method comprising the steps of: [0014] providing a low density
melanocortin 4 receptor (MC4-R) system; [0015] providing a high
density MC4-R system; [0016] determining the functional activity of
the compound in the low density MC4-R system, such as by means of
an adenylyl cyclase expression assay; [0017] determining the
functional activity of the compound in the high density MC4-R
system, such as by means of an adenylyl cyclase expression assay;
[0018] selecting the compound as a candidate if the compound is
functionally inactive in the low MCR-4 density system and is
functionally active in the high density MC4-R system.
[0019] In a related aspect, the low density MC4-R system is a low
density human MC4-R (hMC4-R) system and the high density MC4-R
system is a high density hMC4-R system. In one aspect, the low
density MC4-R system has a melanocortin receptor density determined
by receptor binding saturation that is at least ten times lower
that the melanocortin receptor density in the high density MC4-R
system, at least fifty times lower than the melanocortin receptor
density in the high density MC4-R system or at least one hundred
times lower than the melanocortin receptor density in the high
density MC4-R system.
[0020] In a related aspect, both the low density MC4-R system and
the high density MC4-R system comprise mammalian cells transformed
with a gene encoding for MC4-R. In one aspect, the gene encoding
for MC4-R comprises a gene encoding for hMC4-R. In one aspect, the
gene encoding for MC4-R is under the control of a gene expression
variable regulatory system, for example, a variably inducible
expression system as known in the art. In one aspect, the gene
expression variable regulatory system comprises a
tetracycline-regulated mammalian expression system. In one aspect
the low density MC4-R system employs (receives) at least ten times
less of the inducer of expression, such as tetracycline or a
related regulator, than does the high density MC4-R system. In one
aspect, the low density MC4-R system employs (receives) at least
about one hundred times less inducer of expression, such as
tetracycline or a related regulator, than does the high density
MC4-R system.
[0021] In a related aspect, the low density MC4-R system has a
receptor density expressed as a B.sub.max receptor binding
saturation value using NDP-.alpha.-MSH, such as I.sup.125-labeled
NDP-.alpha.-MSH, of between approximately 80 and 20 fmol/mg or
between approximately 80 and 40 fmol/mg. In a related aspect, the
high density MC4-R system has a receptor density expressed as a
B.sub.max receptor binding saturation value using NDP-.alpha.-MSH,
such as 25-labeled NDP-.alpha.-MSH, of at least about 4,000 fmol/mg
or at least about 6,000 fmol/mg.
[0022] In a related aspect, the compound is functionally inactive
in the low MCR-4 density system where the compound has no
measurable intrinsic activity, or alternatively has an intrinsic
activity of less than about 0.1 (10%), based on maximal stimulation
of adenylyl cyclase achievable by the compound in the low hMC4-R
density system where the maximal stimulation achieved by
.alpha.-MSH or NDP-.alpha.-MSH is designated as an intrinsic
activity of 1.0 (100%).
[0023] In a related aspect, the compound is functionally active in
the high density MC4-R system where the compound has an intrinsic
activity of more than about 0.1 (10%), more than about 0.2 (20%),
more than about 0.3 (30%), more than about 0.5 (50%), or more than
about 0.7 (70%), based on maximal stimulation of adenylyl cyclase
achievable by the compound in the high hMC4-R density system where
the maximal stimulation achieved by .alpha.-MSH or NDP-.alpha.-MSH
is designated as an intrinsic activity of 1.0 (100%).
[0024] In a second aspect, the invention provides a method of
selecting a compound for treating sexual dysfunction, the method
comprising the steps of: [0025] providing a low density MC4-R
system; [0026] providing a high density MC4-R system; [0027]
determining the functional activity of the compound in the low
density MC4-R system, such as by means of an adenylyl cylase
expression assay; [0028] determining the functional activity of the
compound in the high density MC4-R system, such as by means of an
adenylyl cylase expression assay; [0029] selecting the compound as
a candidate for treating sexual dysfunction if the compound is
functionally active in both the low MCR-4 density system and high
density MC4-R system.
[0030] In a related aspect, the method further comprises the steps
of determining the functional status of the compound in a
transfected or transformed MC4-R system not under the control of a
gene expression variable regulatory system and selecting the
compound as a candidate compound further requires that the compound
is an agonist in the transformed MC4-R system not under the control
of a gene expression variable regulatory system. In a related
aspect, the compound is an agonist in the transformed MC4-R system
where the compound has an intrinsic activity of more than about 0.7
(70%), based on maximal stimulation of adenylyl cyclase achievable
by the compound in the transformed hMC4-R system where the maximal
stimulation achieved by .alpha.-MSH or NDP-.alpha.-MSH is
designated as an intrinsic activity of 1.0 (100%).
[0031] In a third aspect, the invention provides a method of
selecting a compound for attenuating food intake and/or for
treating obesity and related energy homeostasis or feeding
disorders characterized by excess weight gain which compound at
least substantially does not induce or initiate a sexual response,
the method comprising the steps of: [0032] providing a low density
MC4-R expression system; [0033] providing a high density MC4-R
expression system; [0034] determining the functional activity of
the compound in an adenylyl cyclase expression assay employing the
low density MC4-R system; [0035] determining the functional
activity of the compound in an adenylyl cyclase expression assay
employing the high density MC4-R system; [0036] selecting the
compound if the compound is functionally inactive in both the low
MCR-4 density system and the high density MC4-R system.
[0037] In a related aspect, the invention further comprises the
step of providing a competition inhibition assay for competitive
binding with .alpha.-MSH as to MC4-R and selecting the compound as
a candidate or further candidate compound further requires that the
compound inhibits at least about 90% of the binding of .alpha.-MSH
to MC4-R. In another related aspect, the invention further
comprises the step of providing an assay for determining the Ki
(nM) of the compound as to .alpha.-MSH binding to MC4-R and
selecting the compound as a candidate or further candidate compound
further requires that the compound has a Ki (nM) of less than about
100, a Ki (nM) of less than about 10, or a Ki (nM) of less than
about 1.
[0038] In a fourth aspect, the invention provides a pharmaceutical
composition for treatment of obesity and/or for attenuating food
intake and body weight gain generally, without substantially
inducing a sexual response, comprising an effective amount of a
melanocortin-4 receptor (MC4-R) specific compound which is
functionally inactive at a concentration of about 1000 nM or less
in an adenylyl cyclase expression assay at a low MC4-R density but
which is functionally active at a concentration of about 1000 nM or
less in an adenylyl cyclase expression assay at a high MC4-R
density, and one or more of carriers, excipients and adjunct
ingredients.
[0039] In a fifth aspect, the invention provides a pharmaceutical
composition for treatment of obesity and/or for attenuating food
intake and body weight gain generally, without substantially
inducing a sexual response, comprising an effective amount of a
melanocortin-4 receptor (MC4-R) specific compound which is
functionally inactive at a concentration of about 1000 nM or less
in an adenylyl cyclase expression assay at a low MC4-R density and
at a high MC4-R density, and one or more of carriers, excipients
and adjunct ingredients.
[0040] In a sixth aspect, the invention provides a pharmaceutical
composition for treatment of sexual dysfunction, comprising an
effective amount of a melanocortin-4 receptor (MC4-R) specific
compound which is functionally active at a concentration of about
1000 nM or less in an adenylyl cyclase expression assay at both a
low MC4-R density and at a high MC4-R density, and one or more of
carriers, excipients and adjunct ingredients.
[0041] One embodiment of the present invention provides
melanocortin receptor-specific agents useful in treatment of
obesity and/or for attenuating food intake and body weight gain
generally, the agents characterized in part in that they doenot
induce a sexual response in a mammal, including not inducing a
penile erection response in a male.
[0042] Another embodiment of the present invention provides a
method for selection of a melanocortin receptor-specific agent
useful in treatment of obesity and/or for attenuating food intake
and body weight gain generally, the method characterized in part in
that agents are selected which have low or no efficacy with respect
to cAMP production in a low density melanocortin receptor
expression system, but which have significant efficacy with respect
to cAMP production in a high density melanocortin receptor
expression system.
[0043] Another embodiment of the present invention provides
melanocortin receptor-specific agents useful in treatment of
obesity and/or for attenuating food intake and body weight gain
generally, the agents characterized in part in that they have
efficacy, such as by cAMP production, equal to or greater than
NDP-.alpha.-MSH in a high density melanocortin receptor expression
system, but less than NDP-.alpha.-MSH, and preferably substantially
less than NDP-.alpha.-MSH, in a low density melanocortin receptor
expression system.
[0044] Another embodiment of the present invention provides a
method for selection of melanocortin receptor-specific agents
useful in treatment of obesity and/or for attenuating food intake
and body weight gain generally, the method characterized in part in
that agents are selected which have low or no efficacy with respect
to cAMP production in a low density hMC4-R (human melanocortin 4
receptor) expression system, but which have significant efficacy
with respect to cAMP production in a high density hMC4-R expression
system.
[0045] Another embodiment of the present invention provides a
method for selection of a melanocortin receptor-specific agent
useful in treatment of obesity and/or for attenuating food intake
and body weight gain generally, the method characterized in part in
that agents are selected which have low or no efficacy with respect
to cAMP production in a low density melanocortin receptor
expression system and in a high density melanocortin receptor
expression system.
[0046] Another embodiment of the present invention provides a
melanocortin receptor-specific agent useful in treatment of obesity
and/or for attenuating food intake and body weight gain generally,
the agent characterized in part in that it is efficacious for
treatment of obesity and/or for attenuating food intake and body
weight gain generally without substantially inducing a sexual
response, such as without substantially inducing a penile erection
response in a male.
[0047] Yet another embodiment of the present invention provides a
melanocortin receptor-specific agent useful in treatment of obesity
and/or for attenuating food intake and body weight gain generally
that binds to MC4-R with high affinity but has low intrinsic
activity in terms of cAMP expression in a low or moderate MC4-R
density expression system.
[0048] Yet another embodiment of the present invention is to
provide a method of treating obesity and/or for attenuating food
intake and body weight gain generally comprising administration of
a melanocortin receptor-specific agent that binds to MC4-R with
high affinity, preferably with a Ki (nM) at MC4-R of about 15 or
less, but has low intrinsic activity in terms of cAMP expression in
a low, moderate or high density MC4-R expression system, and is
efficacious in treating obesity and/or in attenuating food intake
and/or body weight gain.
[0049] Yet another embodiment of the present invention provides a
method of treating obesity and/or for attenuating food intake and
body weight gain generally comprising administration of a
melanocortin receptor-specific agent that has a Ki (nM) at MC4-R,
determined with respect to NDP-.alpha.-MSH, that is half or less
than half, and preferably substantially less than half, of the
EC.sub.50 (nM) for cAMP accumulation at MC4-R in a low or moderate
MC4-R density expression system.
[0050] Yet another embodiment of the present invention provides a
method of treating obesity and/or for attenuating food intake and
body weight gain generally comprising administration of a
melanocortin receptor-specific agent wherein there is at least no
substantial rebound effect, such as no significant immediate
rebound effect, or at least no substantial increase in body weight,
following cessation of administration of the agent. In this
embodiment, the agent at least substantially does not cause an
immediate rebound effect, or stated differently, the agent is not
associated with any substantial rebound effect, particularly no
substantial rebound effect within the first month, or alternatively
within the first two months, following cessation of
administration.
[0051] Yet another embodiment of the present invention provides a
method for selection of a melanocortin receptor-specific agent
useful in treatment of sexual dysfunction, including male erectile
dysfunction and/or female sexual dysfunction, the method
characterized in part in that an agent is selected which has
significant efficacy with respect to cAMP production in a low
hMC4-R density expression system and in a high hMC4-R density
expression system.
[0052] One aspect of this invention provide compounds that bind to
a receptor binding site of .alpha.-MSH, such as on MC4-R,
preferably human MC4-R, with high affinity yet without causing any
activation, or alternatively causing low activation, of adenylyl
cyclase activity in a low density melanocortin receptor expression
system, but which cause activation of adenylyl cyclase in a high
density melanocortin receptor expression system. In another aspect,
the compounds are either inactive or are neutral antagonists with
respect to .alpha.-MSH in low density melanocortin receptor
expression system cAMP stimulation assays. In another aspect, the
compounds are partial agonists in low density melanocortin receptor
systems, but are full agonists in high density melanocortin
receptor systems.
[0053] In another embodiment, the methods of this invention provide
compounds that bind to a receptor binding site of .alpha.-MSH, such
as on MC4-R, preferably hMC4-R, with high affinity and further
cause activation of adenylyl cyclase activity in both a low density
melanocortin receptor expression system and a high density
melanocortin receptor expression system, which compounds may be
employed for treatment of sexual dysfunction, including erectile
dysfunction in males and female sexual dysfunction.
[0054] Other objects, advantages and novel features, and further
scope of applicability of the present invention will be set forth
in part in the detailed description to follow, and in part will
become apparent to those skilled in the art upon examination of the
following, or may be learned by practice of the invention. The
objects and advantages of the invention may be realized and
attained by means of the instrumentalities and combinations
particularly pointed out in the appended claims.
DETAILED DESCRIPTION OF THE INVENTION
[0055] In one broad aspect, the invention describes and discloses
methods for the identification and selection of compounds with
utility for treatment of obesity and related energy homeostasis
diseases, conditions and syndromes, but which compounds do not
induce a sexual response in mammals, including a penile erection
response in males. In a related aspect, the invention describes and
discloses the use of a variety of specified melanocortin
receptor-specific compounds selected by such methods that may be
employed in the treatment of obesity and related energy homeostasis
diseases, conditions and syndromes.
[0056] In one embodiment the invention employs an inducible gene
expression system to transform cells, such as for example HEK-293
cells or CHO cells, with varying receptor densities of hMC4-R. As
used herein, a "transformed" cell system is one constitutively
expressing an MC4-R transgene, most preferably where the system is
stably transformed for constitutive expression of MC4-R. While
preferably the transgene is incorporated into the cell's genome, it
is also possible and contemplated that transfection, including
stable transfection, may be employed. In one inducible gene
expression system using doxycycline as the inducer of gene
expression, the receptor density varies from very low, such as for
example about approximately 70 fmol/mg, to very high, such as for
example about approximately 6300 fmol/mg. By contrast, "normal"
transformed or transfected cells, such as those not employing an
inducible gene expression system, have a receptor density of
approximately about 400 fmol/mg. The invention, in part, relates to
the discovery that MC4-R specific compounds with no efficacy in
terms of cAMP expression in a very low density system, but that
have efficacy (such as equal to or greater than NDP-.alpha.-MSH) in
terms of cAMP expression in a very high density system, are likely
to be effective for treatment of obesity and/or for attenuating
food intake and body weight gain generally without inducing a
sexual response in a mammal, including a penile response in a male
mammal. Conversely, MC4-R specific compounds with efficacy in terms
of cAMP expression in both a very low density system and a very
high density system, are likely to be effective for treatment of
sexual dysfunction, include male erectile dysfunction and female
sexual disorder. Without wishing to be bound by theory, it is one
hypothesis of the inventors that hMC4-R in very low density
expressing cells have a different conformation than hMC4-R in very
high density expressing cells. Thus, by way of hypothesis only,
G-protein activation may be lower in low density cells unless the
ligand induces a change in the conformation of the receptors;
alternatively, and again by way of hypothesis only, very low
density hMC4-R may be in one or more inactive receptor states,
while high or very high density hMC4-R are in one or more active
receptor states. It is further hypothesized, again without wishing
to be bound by theory, that the observed difference may be due to
one or more factors such as receptor internalization,
desensitization, oligomerization, phosphorylation or association
with other membrane proteins.
[0057] In the practice of a method of this invention, a
melanocortin receptor-specific agent, and preferably an agent
specific for MC4-R, is selected, which agent is characterized in
that: (a) the agent attenuates the binding of
alph.alpha.-melanocyte stimulating hormone (.alpha.-MSH) to
melanocortin receptors; (b) the agent has a low efficacy, such that
the agent is a weak agonist, weak antagonist, neutral antagonist,
inverse agonist or protean agonist at MC4-R, and preferably has an
efficacy or intrinsic activity in terms of cAMP expression of less
than about 50%, more preferably less than about 30%, and most
preferably less than about 10% of that of .alpha.-MSH in a low
density MC4-R expression system; and, (c) optionally and
preferably, the agent is further characterized in that the agent
has an efficacy in terms of cAMP expression of greater than about
50%, more preferably greater than about 80%, and most preferably
equal to or greater than about 100% of that of .alpha.-MSH in a
high density MC4-R expression system and an efficacy or intrinsic
activity in terms of cAMP expression of less than about 50%, more
preferably less than about 30%, and most preferably less than about
10% of that of .alpha.-MSH in a low density MC4-R expression
system.
[0058] The melanocortin receptor-specific agents disclosed herein,
and the methods for selecting such agents, are further
characterized in that the agents in general do not significantly
induce a sexual response in a mammal, such as a penile erection
response in a male. A number of melanocortin-receptor specific
compounds and agents heretofore evaluated for use in treatment of
obesity and related conditions have had unacceptable side effects
relating to initiation or induction of a sexual response, including
a penile erection response in males. In part, compounds of the
invention, and compounds selected by methods of the invention, do
not generally induce a sexual response.
[0059] The methods disclosed herein may be employed with any
compound believed or suspected to bind to a melanocortin receptor,
preferably to bind to MC4-R, and more preferably to bind to hMC4-R.
Thus the methods and this invention may be employed with peptide,
peptidomimetic, metallopeptide, small molecule, ring-core small
molecule and other compounds known in the art, including compounds
hereafter developed. While the invention has been exemplified with
the compounds disclosed herein, the invention is not limited to
such compounds, and may be applied to other compounds, which
compounds may significantly differ from the compounds disclosed
herein.
[0060] With respect to the compounds disclosed herein, the methods
of synthesis and purification, methods of testing, formulas and
definitions of classes of compounds and specific compounds
disclosed in the following patent applications are relevant to the
practice of the invention: U.S. patent application Ser. No.
11/557,408, entitled N-Alkylated Cyclic Peptide Melanocortin
Agonists, filed on Nov. 7, 2006; U.S. patent application Ser. No.
11/464,069, entitled Melanocortin Receptor-Specific piperazine and
Keto-piperazine Compounds, filed on Aug. 11, 2006; U.S. patent
application Ser. No. 11/464,051, entitled Substituted Melanocortin
Receptor-Specific Single Acyl piperazine Compounds, filed on Aug.
11, 2006; U.S. patent application Ser. No. 11/464,053, entitled
Melanocortin Receptor-Specific piperazine Compounds with Diamine
Groups, filed on Aug. 11, 2006; U.S. patent application Ser. No.
11/110,060, entitled Substituted Melanocortin Receptor-Specific
piperazine Compounds, filed on Apr. 19, 2005; U.S. patent
application Ser. No. 11/099,814, entitled Substituted Melanocortin
Receptor-Specific piperazine Compounds, filed on Apr. 5, 2005; U.S.
patent application Ser. No. 11/040,838, entitled
Thieno[2,3-d]Pyrimidine-2,4-Dione Melanocortin-Specific Compounds,
filed on Jan. 21, 2005; U.S. patent application Ser. No.
11/036,282, entitled Naphthlalene-Containing Melanocortin
Receptor-Specific Small Molecules, filed on Jan. 14, 2005; U.S.
patent application Ser. No. 10/837,519, entitled Melanocortin
Receptor-Specific Compounds, filed on Apr. 30, 2004; U.S. patent
application Ser. No. 10/762,079, entitled piperazine
Melanocortin-Specific Compounds, filed on Jan. 21, 2004, and issued
as U.S. Pat. No. 7,354,923 on Apr. 8, 2008; U.S. patent application
Ser. No. 10/761,889, entitled Bicyclic Melanocortin-Specific
Compounds, filed on Jan. 21, 2004, and issued as U.S. Pat. No.
7,326,707 on Feb. 5, 2008; International Patent Application
PCT/US02/22196, entitled Linear and Cyclic Melanocortin
Receptor-Specific Peptides, filed on Nov. 7, 2002; International
Patent Application PCT/US02/25574, entitled Peptidomimetics of
Biologically Active Molecules, filed on Aug. 12, 2002; and
International Patent Application PCT/US01/50075, entitled
Identification of Target-Specific Folding Sites in Peptides and
Proteins, filed on Dec. 19, 2001. The specification and claims of
each of the foregoing patent applications is incorporated herein by
reference.
[0061] Definitions. Before proceeding further with the description
of the invention, certain terms are defined as set forth
herein.
[0062] The "amino acid" and "amino acids" used in this invention,
and the terms as used in the specification and claims, include the
known naturally occurring protein amino acids, which are referred
to by their common three letter abbreviation. See generally
Synthetic Peptides: A User's Guide, G A Grant, editor, W.H. Freeman
& Co., New York, 1992, the teachings of which are incorporated
herein by reference, including the text and table set forth at
pages 11 through 24. Conventional amino acid residues have their
conventional meaning as given in Chapter 2400 of the Manual of
Patent Examining Procedure, 8.sup.th Ed. Thus, "Nle" is norleucine,
"Asp" is aspartic acid, "His" is histidine, "D-Phe" is
D-phenylalanine, "Arg" is arginine, "Trp" is tryptophan, "Lys" is
lysine, "Gly" is glycine, "Pro" is proline, "Tyr" is tyrosine,
"Ser" is serine and so on.
[0063] The term "composition", as in pharmaceutical composition, is
intended to encompass a product comprising the active
ingredient(s), and the inert ingredient(s) that make up the
carrier, as well as any product which results, directly or
indirectly, from combination, complexation or aggregation of any
two or more of the ingredients, or from dissociation of one or more
of the ingredients, or from other types of reactions or
interactions of one or more of the ingredients. Accordingly, the
pharmaceutical compositions of the present invention encompass any
composition made by admixing a compound of the present invention
and one or more pharmaceutically acceptable carriers, and
optionally one or more pharmaceutically active ingredients and
agents.
[0064] A variety of amino acids, chemicals and compounds are
employed in this invention, and the following abbreviations have
the meanings given:
[0065] Ac acetyl group CH.sub.3CO--
[0066] Boc tertiary butyloxycarbonyl
[0067] Cbz benzyloxycarbonyl
[0068] DMF N,N-dimethylformamide
[0069] DMSO dimethylsulfoxide
[0070] Fmoc 9-fluorenylmethoxycarbonyl
[0071] HEPES 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid
[0072] Heptanoyl CH.sub.3--(CH.sub.2).sub.5CO--
[0073] Nal 1 3-(1-naphthyl)alanine
[0074] Nal 2 3-(2-naphthyl)alanine
[0075] 2-Naphthylacetyl 2-naphthyl-CH.sub.2CO--
[0076] Phe(4-Cl) 4-chloro-phenylalanine
[0077] Phe(2,4-diCl) 2,4,-dichloro-phenylalanine
[0078] Prt A protecting group, such as Boc, Cbz or Fmoc
[0079] Ser(Bzl) O-benzyl-serine
[0080] Thr(Bzl) O-Benzyl-threonine
[0081] By a melanocortin receptor "agonist" is meant an endogenous
or drug substance or compound, including a compound of this
invention, that can interact with a melanocortin receptor and
initiate a pharmacological response, including but not limited to
an ascertainable functional activity, for example adenylyl cyclase
activation, characteristic of the melanocortin receptor. By a
melanocortin receptor "antagonist" is meant a drug or a compound,
including a compound of this invention, that opposes the
melanocortin receptor-associated responses normally induced by a
melanocortin receptor agonist agent, but without itself initiating
a pharmacological response characteristic of the melanocortin
receptor, such as increasing or decreasing adenylyl cyclase
activation. By a melanocortin receptor "inverse agonist" is meant a
drug or a compound, including a compound of this invention, which
is an antagonist with respect to an agonist, and which further by
itself induces or initiates a pharmacological response
characteristic of the melanocortin receptor, such as reducing basal
or constitutive adenylyl cyclase activation. By a melanocortin
receptor "protean agonist" is meant a drug or a compound, including
a compound of this invention, which acts as either an inverse
agonist or an agonist, depending on the constitutive activity of
the MC4-R, either promoting a switch to a less active conformation
or enriching the active conformation. In general, inverse and
protean agonists are discussed at length in Kenakin, T. Inverse,
protean, and ligand-selective agonism: matters of receptor
conformation. The FASEB Journal, 15:598-611, 2001, incorporated
here by reference as if set forth in full.
[0082] By ".alpha.-MSH" is meant the peptide
Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH.sub.2
(SEQ ID NO:1) and analogs and homologs thereof, including without
limitation NDP-.alpha.-MSH.
[0083] By "NDP-.alpha.-MSH" is meant the peptide
Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH.sub.2
and analogs and homologs thereof.
[0084] A compound that attenuates binding is a compound or drug
prevents or inhibits the binding of .alpha.-MSH to melanocortin
receptors, and preferably MC4-R, or decreases the binding affinity
of .alpha.-MSH to melanocortin receptors, and preferably MC4-R.
Compounds of this invention attenuate the binding of .alpha.-MSH to
melancortin receptors. For attenuation of .alpha.-MSH binding, it
is preferable if the compound being tested inhibits at least about
70% of .alpha.-MSH binding, such as iodinated NDP-.alpha.-MSH
binding in a competitive inhibition assay, and more preferably
inhibits at least about 85% of .alpha.-MSH, and most preferably
inhibits at least about 95% or greater of .alpha.-MSH. The percent
inhibition of binding can be readily determined by those skilled in
the art by competition and other inhibition assays, including the
methods disclosed herein and other comparable methods.
[0085] By "EC.sub.50" is meant the molar concentration of an
agonist, including a partial agonist, which produced 50% of the
maximum possible response for that agonist. By way of example, a
test compound which, at a concentration of 72 nM, produces 50% of
the maximum possible response for that compound as determined in a
cAMP assay in an MC4-R cell expression system has an EC.sub.50 of
72 nM. Unless otherwise specified, the molar concentration
associated with an EC.sub.50 determination is in nanomoles per
liter (nM).
[0086] By "Ki (nM)" is meant the equilibrium inhibitor dissociation
constant representing the molar concentration of a competing
compound that binds to half the binding sites of a receptor at
equilibrium in the absence of radioligand or other competitors. In
general, the numeric value of the Ki is inversely correlated to the
affinity of the compound for the receptor, such that if the Ki is
low, the affinity is high. Ki may be determined using the equation
of Cheng and Prusoff (Cheng Y., Prusoff W. H., Biochem. Pharmacol.
22: 3099-3108, 1973):
K i = EC 50 1 + [ ligand ] K D ##EQU00001##
where "ligand" is the concentration of radioligand and K.sub.D is
an inverse measure of receptor affinity for the radioligand which
produces 50% receptor occupancy by the radioligand. Unless
otherwise specified, the molar concentration associated with a Ki
determination is in nM. Ki may be expressed in terms of specific
receptors (e.g., MC1-R, MC3-R, MC4-R or MC5-R) and specific ligands
(e.g., .alpha.-MSH).
[0087] By "inhibition" is meant the percent attenuation, or
decrease in receptor binding, in a competitive inhibition assay
compared to a known standard. Thus, by "inhibition at 1 .mu.M
(NDP-.alpha.-MSH)" is meant the percent decrease in binding of
NDP-.alpha.-MSH by addition of a determined amount of the compound
to be tested, such as 1 .mu.M of a test compound, such as under the
assay conditions hereafter described. By way of example, a test
compound that does not inhibit binding of NDP-.alpha.-MSH has a 0%
inhibition, and a test compound that completely inhibits binding of
NDP-.alpha.-MSH has a 100% inhibition. Typically, as described
hereafter, a radio assay is used for competitive inhibition
testing, such as with I.sup.125-labeled NDP-.alpha.-MSH. However,
other methods of testing competitive inhibition are known,
including use of label or tag systems other than radioisotopes, and
in general any method known in the art for testing competitive
inhibition may be employed in this invention. It may thus be seen
that "inhibition" is one measure to determine whether a test
compound attenuates binding of .alpha.-MSH to melanocortin
receptors.
[0088] By "pA.sub.2" is meant a logarithmic measure of the potency
of a compound that is an antagonist, including a partial
antagonist, or the negative log of the concentration of the
antagonist which produces a 2-fold shift in the
concentration-response curve for an agonist. The pA.sub.2 value is
typically determined by the intercept on the y-axis of the
extrapolated line in a Schild plot plotting the log (concentration
ratio--1) against the log (antagonist concentration). See generally
Schild, H. O. pA, a new scale for the measurement of drug
antagonism. Br. J. Pharmacol. 2:189-206, 1947; and Arunlakshana, O.
and Schild, H. O. Some quantitative uses of drug antagonists. Br.
J. Pharmacol. 14:48-58, 1949.
[0089] By "binding affinity" is meant the ability of a compound or
drug to bind to its biological target, expressed herein as Ki
(nM).
[0090] By "intrinsic activity" is meant the maximal functional
activity achievable by a compound in a specified melanocortin
receptor expressing cell system, such as the maximal stimulation of
adenylyl cyclase. The maximal stimulation achieved by .alpha.-MSH
or NDP-.alpha.-MSH is designated as an intrinsic activity of 1.0
(or 100%) and a compound capable of stimulating half the maximal
activity that of .alpha.-MSH or NDP-.alpha.-MSH is designated as
having an intrinsic activity of 0.5 (or 50%). A compound of this
invention that under assay conditions described herein has an
intrinsic activity of 0.7 (70%) or higher is classified as an
agonist, a compound with intrinsic activity between 0.1 (10%) and
0.7 (70%) is classified as a partial agonist, and a compound with
intrinsic activity below 0.1 (10%) is classified as inactive or
having no intrinsic activity. The intrinsic activity may be
determined with respect to a low density melanocortin receptor
expression system, a high density melanocortin receptor expression
system, or a melanocortin receptor expression system which is not
made, designed or selected so as to be either a low density or high
density expression system.
[0091] In general, "functional activity" is a measure of the
signaling of a receptor, or measure of a change in the receptor
associated with signaling of the receptor, such as a melanocortin
receptor, and in particular MC4-R or hMC4-R, upon activation by a
compound. Melanocortin receptors initiate signal transduction
through activation of heterotrimeric G proteins. In one aspect,
melanocortin receptors signal through G.alpha..sub.S, which
catalyzes production of cAMP by adenylyl cyclase. Thus
determination of stimulation of adenylyl cyclase, such as
determination of maximal stimulation of adenylyl cyclase, is one
measure of functional activity, and is the primary measure
exemplified herein. However, it is to be understood that
alternative measures of functional activity may be employed in the
practice of this invention, and are specifically contemplated and
included within the scope of this invention. Thus, in one example
intracellular free calcium may be measured, such as reported by and
using the methods disclosed in Mountjoy K. G. et al., Melanocortin
receptor-medicated mobilization of intracellular free calcium in
HEK293 cells. Physiol Genomics 5:11-19, 2001, or Kassack M. U. et
al., Functional screening of G protein-coupled receptors by
measuring intracellular calcium with a fluorescence microplate
reader. Biomol Screening 7:233-246, 2002. It is also possible to
measure activation by measurement of the production of inositol
triphosphate or diacylglycerol from phosphatidylinositol
4,5-biphosphate, such as by use of radioassays. Yet another measure
of functional activity is receptor internalization, resulting from
activation of regulatory pathways, such as using the methods
disclosed in Nickolls S. A. et al., Functional selectivity of
melanocortin 4 receptor peptide and nonpeptide agonists: evidence
for ligand specific conformational states. J Pharm Exper
Therapeutics 313:1281-1288, 2005. Yet another measure of functional
activity is the exchange, and exchange rate, of nucleotides
associated with activation of a G protein receptor, such as the
exchange of GDP (guanosine diphosphate) for GTP (guanosine
triphosphase) on the G protein .alpha. subunit, which may be
measured by any number of means, including a radioassay using
guanosine 5'-(.gamma.-[.sup.35S]thio)-triphosphate, as disclosed in
Manning D. R., Measures of efficacy using G proteins as endpoints:
differential engagement of G proteins through single receptors. Mol
Pharmacol 62:451-452, 2002. Various gene-based assays have been
developed for measuring activation of G-coupled proteins, such as
those disclosed in Chen W. et al., A calorimetric assay from
measuring activation of Gs- and Gq-coupled signaling pathways. Anal
Biochem 226:349-354, 1995; Kent T. C. et al., Development of a
generic dual-reporter gene assay for screening G-protein-coupled
receptors. Biomol Screening, 5:437-446, 2005; or Kotarsky K. et
al., Improved receptor gene assays used to identify ligands acting
on orphan seven-transmembrane receptors. Pharmacology &
Toxicology 93:249-258, 2003. The calorimetric assay of Chen et al.
has been adapted for use in measuring melanocortin receptor
activation, as disclosed in Hruby V. J. et al., Cyclic lactam
.alpha.-melanocortin analogues of Ac-Nle.sup.4-cyclo[Asp.sup.5,
D-Phe.sup.7, Lys.sup.10] .alpha.-melanocyte-stimulating
hormone-(4-10)-NH.sub.2 with bulky aromatic amino acids at position
7 shows high antagonist potency and selectivity at specific
melanocortin receptors. J Med Chem 38:3454-3461, 1995. In general,
functional activity may be measured by any method, including
methods of determining activation and/or signaling of a G-coupled
receptor, and further including methods which may be hereafter
developed or reported. Each of the foregoing articles, and the
methods disclosed therein, is incorporated here by reference as if
set forth in full.
[0092] Clinical Applications. The compounds disclosed herein and/or
selected by the methods disclosed herein can be used for both
medical applications and animal husbandry or veterinary
applications. Typically, the product is used in humans, but may
also be used in other mammals. The term "patient" is intended to
denote a mammalian individual, and is so used throughout the
specification and in the claims. The primary applications of this
invention involve human patients, but this invention may be applied
to laboratory, farm, zoo, wildlife, pet, sport or other
animals.
[0093] In the practice of the method of this invention, compounds
may be employed that are MC4-R agonists, partial agonists,
antagonists, inverse agonists, or protean agonists functionally
inactive in low density MC4-R expression systems, yet with
demonstrated efficacy in animal models in modifying energy
metabolism and feeding behavior. Thus, in one embodiment the
compounds are inactive or have no intrinsic activity with respect
to MC4-R at low densities, but bind MC4-R with high affinity and,
in some instances, selectivity, and further have demonstrated
efficacy, in animal models, in modifying energy metabolism and
feeding behavior.
[0094] Formulations. The compounds may be formulated by any means
known in the art, including but not limited to tablets, capsules,
caplets, suspensions, powders, lyophilized forms and
aerosols/aerosolizable forms and may be mixed and formulated with
buffers, binders, stabilizers, anti-oxidants and other agents known
in the art. The compounds may be administered by any systemic or
partially systemic means such as those known in the art, including
but not limited to intravenous injection, subcutaneous injection,
administration through mucous membranes, oral administration,
dermal administration, skin patches, aerosols and the like.
[0095] The invention further provides a pharmaceutical composition
and method for the use thereof that includes a compound of this
invention and a pharmaceutically acceptable carrier. The compounds
may thus be formulated or compounded into pharmaceutical
compositions that include at least one compound of this invention,
or a compound selected by a method of this invention, together with
one or more pharmaceutically acceptable carriers, including
excipients, such as diluents, carriers and the like, and additives,
such as stabilizing agents, preservatives, solubilizing agents,
buffers and the like, as may be desired. Formulation excipients may
include polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,
polyethylene glycol, mannitol, sodium chloride or sodium citrate.
For injection or other liquid administration formulations, water
containing at least one or more buffering constituents is suitable,
and stabilizing agents, preservatives and solubilizing agents may
also be employed. For solid administration formulations, any of a
variety of thickening, filler, bulking and carrier additives may be
employed, such as starches, sugars, fatty acids and the like. For
pharmaceutical formulations, it is also contemplated that any of a
variety of measured-release, slow-release or time-release
formulations and additives may be employed, such that the dosage
may be formulated so as to effect delivery of a compound of this
invention over a period of time.
[0096] The compounds of this invention, including compounds not
disclosed herein but selected by a method of this invention, may be
in the form of any pharmaceutically acceptable salt. Acid addition
salts of the compounds of this invention are prepared in a suitable
solvent from the compound and an excess of an acid, such as
hydrochloric, hydrobromic, sulfuric, phosphoric, acetic,
trifluoroacetic, maleic, succinic or methanesulfonic acids. The
acetate salt form is especially useful. Where the compounds of this
invention include an acidic moiety, suitable pharmaceutically
acceptable salts may include alkali metal salts, such as sodium or
potassium salts, or alkaline earth metal salts, such as calcium or
magnesium salts.
[0097] The compounds and pharmaceutical compositions may be
administered by injection, which injection may be intravenous,
subcutaneous, intramuscular, intraperitoneal or by any other means
known in the art. In general, any route of administration by which
the compounds of this invention are introduced across an epidermal
layer of cells may be employed. Administration means may include
administration through mucous membranes, buccal administration,
oral administration, dermal administration, inhalation
administration, nasal administration and the like. The dosage for
treatment is administration, by any of the foregoing means or any
other means such as those known in the art, of an amount sufficient
to bring about the desired therapeutic effect.
[0098] Therapeutically Effective Amount. In general, the actual
quantity of compound administered to a patient will vary between
fairly wide ranges depending upon the mode of administration, the
formulation used, and the response desired. This may readily be
determined by one of ordinary skill in the art through means such
as pharmacokinetic studies, plasma half-life studies, dose
escalation studies, and the like. The dosage for treatment is
administration, by any of the foregoing means or any other means
known in the art, of an amount sufficient to bring about the
desired therapeutic or biological effect. Thus, a therapeutically
effective amount includes an amount of a compound or pharmaceutical
composition of this invention that is sufficient to induce the
desired therapeutic or biological effect.
[0099] The compounds of this invention are highly active in
modifying energy metabolism and feeding behavior. For example, the
compound can be administered at 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50,
100, or 500 .mu.g/kg body weight, depending on the specific
compound selected, the desired therapeutic response, the route of
administration, the formulation and other factors known to those of
skill in the art. Conventional dose response studies and other
pharmacological means may be employed to determine the optimal dose
for a desired effect with a given compound, given formulation and
given route of administration.
[0100] Combination Therapy and Weight Regulation. It is also
possible and contemplated to use compounds of this invention, or
compounds selected by a method of this invention, in combination
with other drugs or agents for treatment of various weight and
feeding-related disorders. Any of the combinations disclosed in
U.S. patent application Ser. No. ______ (Attorney Docket No.
056291-5365), entitled Compounds and Methods for Treating Obesity,
which is filed concurrently herewith and which claims priority to
U.S. Provisional Patent Application Ser. No. 60/712,282, may be
employed here, and the teachings thereof are specifically
incorporated herein by reference.
Assays and Animal Models.
[0101] Selected compounds were tested in assays to determine
binding and functional status, and were tested in animal models of
penile erection and feeding behavior, as discussed below. The
following assays and animal models were employed, with
modifications as discussed in the examples.
[0102] Competitive inhibition assay using
[I.sup.125]-NDP-.alpha.-MSH. A competitive inhibition binding assay
was conducted using membranes prepared from hMC3-R, hMC4-R, hMC5-R,
and B-16 mouse melanoma cells (containing MC1-R) in 25 mM HEPES
buffer (pH 7.5) with 100 mM NaCl, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2,
0.3 mM 1,10-phenanthroline, and 0.2% bovine serum albumin, and the
radiolabeled ligand [I.sup.125]-NDP-.alpha.-MSH (New England
Nuclear). The ligand concentration used was 0.4 nM for MC3-R and
MC5-R, 0.2 nM for MC4-R and 0.1 nM for B16-F10 (MC1-R) membranes.
In certain experiments, hMC1-R was also employed. The assay tube
also contained a chosen concentration of the test compound of this
invention, typically a 1 .mu.M concentration, for determining its
efficacy in inhibiting the binding of [I.sup.125]-NDP-.alpha.-MSH
to its receptor. Non-specific binding was measured by inhibition of
binding of [I.sup.125]-NDP-.alpha.-MSH in the assay with the
presence of 1 .mu.M .alpha.-MSH.
[0103] The assay mixture was incubated for 90 minutes at room
temperature, then filtered and the membranes washed three times
with ice cold buffer. The filter was dried and counted in a gamma
counter for remaining radioactivity bound to the membranes. 100%
specific binding was defined as the difference in radioactivity
(cpm) bound to cell membranes in the absence and presence of 1
.mu.M .alpha.-MSH. The cpm obtained in presence of test compounds
were normalized with respect to 100% specific binding to determine
the percent inhibition of [I.sup.125]-NDP-.alpha.-MSH binding. Each
assay was conducted in triplicate and the actual mean values are
described, with results less than 0% reported as 0%.
[0104] High and Low Receptor Density Systems. A
tetracycline-regulated mammalian expression system that uses
regulatory elements from the E. coli Tn 10-encoded tetracycline
(Tet) resistance operon was employed (T-REx.TM. System,
Invitrogen). The T-REx.TM. System Manual, published by Invitrogen,
is incorporated by reference. By use of the T-REx.TM. System,
expression of the gene of interest, the human MC4-R gene, was
repressed in the absence of tetracycline or doxycycline and induced
in the presence of tetracycline or doxycycline, with receptor
density being dependent on the concentration of tetracycline or
doxycycline. See generally Yao F. et al: Tetracycline Repressor,
tetR, Rather than the tetR-Mammalian Cell Transcription Factor
Fusion Derivatives, Regulates Inducible Gene Expression in
Mammalian Cells. Hum. Gene Ther. 9:1939-1950 (1998), incorporated
here by reference. Regulation was based on the binding of
tetracycline or doxycycline, a derivative of tetracycline, to the
Tet repressor and derepression of the promoter controlling
expression of the human MC4-R gene. In general tetracycline or
related regulators, such as tetracycline-derivatives, may be used
to induce the gene expression. An inducible expression plasmid for
expression of human MC4-R gene under the control of the strong
human cytomegalovirus immediate-early (CMV) promoter and two
tetracycline operator 2 (TetO.sub.2) sites was employed, together
with a regulatory plasmid, pcDNA6/TR, which encodes the Tet
repressor (TetR) under the control of the human SV40 promoter.
Thus, expression of the human MC4-R gene from the inducible
expression vector was controlled by the strong CMV promoter into
which 2 copies of the tet operator 2 (TetO.sub.2) sequence had been
inserted in tandem. The TetO.sub.2 sequences consisted of 2 copies
of the 19 nucleotide sequence, 5'-TCCCTATCAGTGATAGAGA-3' (SEQ ID
NO: 2) separated by a 2 base pair spacer. Each 19 nucleotide
TetO.sub.2 sequence served as the binding site for 2 molecules of
the Tet repressor. The expression vector pcDNA4/TO/MC4R was
introduced into TREx-293 (Invitrogen, R710-07), a cell line stably
expressing the tetracycline repressor pcDNA6/TR using lipofectamin
2000 reagent (Invitrogen, 11668-019). Alternatively, both the
expression vector and repressor are introduced into host cells by
standard transformation or transfection methods.
[0105] The receptor density at different doxycycline concentrations
was quantified in terms of binding per mg of protein derived from
cell membranes using [I.sup.125]-NDP-.alpha.-MSH in receptor
binding saturation studies to determine a B.sub.max value. Table 1
below quantifies the receptor density:
TABLE-US-00001 TABLE 1 Doxycycline B.sub.max .+-. SD K.sub.D(app)
.+-. SD (ng/mL) (fmol/mg) (pM) n 0.1 (62 .+-. 13) (114 .+-. 57) 8 1
207 .+-. 128 118 .+-. 37 11 10 10113 .+-. 2538 139 .+-. 41 11
[0106] By comparison, transformed or transfected cells not under
control of a tetracycline- or doxycycline-regulated expression
system, such as cells used under the heading "Competitive
inhibition assay using [I.sup.125]-NDP-.alpha.-MSH" above and
elsewhere in this application, typically resulted in a B.sub.max
value of approximately 400 fmol/mg using
[I.sup.125]-NDP-.alpha.-MSH.
[0107] In other studies, up to 100 ng/mL of doxycycline was
employed. However, analysis of data showed that the difference
between 0.1 and 10 ng/mL of doxycycline was sufficient to
differentiate binding as hereafter described.
[0108] In general, any cell line which may be reproducibly
transformed or transfected, stably or transiently, may be employed.
Thus, conventional mammalian cell lines, such as HEK-293 and CHO
(Chinese hamster ovary) cell lines may readily be employed.
Similarly, any controlled expression system may be employed, and
lines that express high and low densities of receptors selected.
Conventionally such expression systems are under the control of a
drug, such as an antibiotic such as tetracycline. However, any
expression system, such as those known in the art, which is able to
reproducibly vary the expression level of receptors may be employed
in the practice of the invention, and it is intended to encompass
all such methods, cells, and like within the scope of the
invention. It will be apparent to those skilled in the art that
transformation, transient transfection or stable transfection or a
combination thereof may be employed to express the necessary
cell-expressed components of the assays of the invention, including
the melanocortin receptor, within the cells which are preferably
but not necessarily mammalian cells. Thus, for example, the cells
of the assay may comprise a transgene encoding and permitting
expression, variable or constitutive as needed, of the melanocortin
receptor. The transgene may be stably integrated in the genome of
the cells or may be present on a transient expression vector. In
one alternative, an endogenous melanocortin receptor gene is placed
under control of inducible gene regulatory elements by stably
introducing the gene elements into a cell such that they are
operably linked to the endogenous melanocortin receptor gene.
[0109] In an alternative embodiment, any selection method may be
employed in a population of cells that are transformed or
transfected to express a melanocortin receptor, with cells
expressing low densities and high densities selected. Selection,
particularly over successive generations, may result in cell lines
which express the desired densities, and are sufficiently stable to
permit reproducible experimentation.
[0110] As used in the specification and claims, a "low density
melanocortin receptor system" includes, but is not limited to, a
transfected or transformed cell gene expression system, preferably
under the control of a gene expression variable regulatory system
such as described above, which has a melanocortin receptor density
measured in receptor binding saturation experiments that is at
least ten times lower, and preferably is more than ten times lower,
such as about 50 to 100 times lower, than the melanocortin receptor
density in a "high density melanocortin receptor system." A "high
density melanocortin receptor system" includes, but is not limited
to, a transfected or transformed cell gene expression system,
preferably under the control of a gene expression variable
regulatory system such as described above.
[0111] A "low density MC4-R system" is a low density melanocortin
receptor system wherein the melanocortin receptor is MC4-R,
preferably human MC4-R (hMC4-R). A "high density MC4-R system" is a
high density melanocortin receptor system wherein the melanocortin
receptor is MC4-R, preferably hMC4-R.
[0112] A transformed cell system not employing a variable
regulatory system, such as transformation of HEK-293 cells with a
human MC4-R gene, may also be employed in the practice of the
invention, and as such is neither a low density melanocortin
receptor system nor a high density melanocortin receptor system.
Any suitable transformation or transfection methodology, including
without limitation those for stable transfection and those for
transient transfection, or reagents may be used, such as use of
Polyfect (QIAGEN, Valencia, Calif.) generally following the
manufacturer's instructions or as described by Gantz I., Miwa H.,
et al. Molecular cloning, expression, and gene localization of a
fourth melanocortin receptor. J. Biol. Chem. 268:15174-15179. 1993.
Such a transformed system is called herein a "normal melanocortin
receptor density system," and is "normal" in that the system is
transformed at the usual rate for the transformed methodology and
reagents employed, and is not under the control of a gene
expression variable regulatory system.
[0113] In a preferred embodiment of the present invention, a low
density melanocortin receptor system has a receptor density, such
as density of hMC4-R, which is lower than receptor density
typically present in a normal melanocortin receptor density system.
Preferably the receptor density is between about 5 and 20 times
lower, more preferably between about 5 and 10 times lower. It may
thus be seen that if a normal melanocortin receptor density system,
such as HEK-293 cells transfected or transformed with a human MC4-R
gene, has a receptor density expressed as a B.sub.max value of
approximately 400 fmol/mg using [I.sup.125]-NDP-.alpha.-MSH in
receptor binding saturation experiments, that a low density
melanocortin receptor system transformed with a human MC4-R gene
would have a receptor density expressed as a B.sub.max value of
between approximately 80 and 20 fmol/mg using
[I.sup.125]-NDP-.alpha.-MSH, preferably between approximately 80
and 40 fmol/mg using [112]-NDP-.alpha.-MSH. Thus, a system having
cells with a B.sub.max.+-.SEM (fmol/mg) of about 62.+-.13, as used
herein, is a low density melanocortin receptor system using
hMC4-R.
[0114] In a related preferred embodiment of the present invention,
a high density melanocortin receptor system has a receptor density,
such as density of MC4-R, which is higher than receptor density in
a normal melanocortin receptor density system. Preferably the
receptor density is between about 10 and 30 times higher, more
preferably between about 10 and 25 times higher. However, an even
higher receptor density may be employed. It may thus be seen that
if a normal melanocortin receptor density system, such as HEK-293
cells transfected or transformed with a human MC4-R gene, has a
receptor density expressed as a B.sub.max value of approximately
400 fmol/mg using [I.sup.125]-NDP-.alpha.-MSH, that a high density
melanocortin receptor system transformed with a human MC4-R gene
would have a receptor density expressed as a B.sub.max value of
between approximately 4,000 and 12,000 fmol/mg using
[I.sup.125]-NDP-.alpha.-MSH in receptor binding saturation
experiments, preferably between approximately 4,000 and 10,000
fmol/mg using [I.sup.125]-NDP-.alpha.-MSH. Thus, a system having
cells with a B.sub.max.+-.SEM (fmol/mg) of about 10,113.+-.2538, as
used herein, is a high density melanocortin receptor system using
hMC4-R.
[0115] It may further be seen that the low density melanocortin
receptor system using hMC4-R expressing cells with a
B.sub.max.+-.SEM (fmol/mg) of about 62.+-.13 has a receptor density
that is about 163 times (ranging from about 101 to 258 times) lower
than the receptor density of the high density melanocortin receptor
system using hMC4-R expressing cells with a B.sub.max.+-.SEM
(fmol/mg) of about 10,113.+-.2538.
[0116] It is to be understood that in biological cell gene
expression systems as described herein there is inherently some
drift in both the density of receptors and responsiveness of the
systems. As is known, even normal melanocortin receptor density
system cells may have to be re-established on a regular basis to
maintain a reasonably constant response. Additionally, where
relative efficacy is to a biologically active peptide, it is
possible and known that relative efficacy compared to such peptide,
such as relative efficacy compared to NDP-.alpha.-MSH, may
fluctuate from one preparation to another on different days, but
generally without change in the rank order for a set of test
compounds.
[0117] General method for EC.sub.50 determination in functional
activity assay. Functional evaluation of compounds at melanocortin
receptors was performed by measuring the accumulation of
intracellular cAMP in HEK-293 cells expressing human MC3-R, MC4-R
or MC5-R, and in B-16 mouse melanoma cells expressing MC1-R. Cells
suspended in Earle's Balanced Salt Solution containing 10 mM HEPES
(pH 7.5), 5 mM MgCl.sub.2, 1 mM glutamine, 0.1% albumin and 0.6 mM
3-isobutyl-1-methyl-xanthine, a phosphodiesterase inhibitor, were
plated in 96 well plates at a density of 0.5.times.10.sup.5 cells
per well. Cells were incubated with the test compounds in the
presence or absence of .alpha.-MSH for 1 hour at 37.degree. C. cAMP
levels in the cell lysates were measured using an EIA kit
(Amersham). Data analysis and EC.sub.50 values were determined
using nonlinear regression analysis with Prism Graph-Pad
software.
[0118] Functional status. The agonist/antagonist status with
respect to MC1-R, MC-3R, MC4-R and MC5-R of certain compounds of
the invention was determined. Antagonistic activity was determined
by measuring the inhibition of .alpha.-MSH-induced or
NDP-.alpha.-MSH-induced cAMP levels following exposure to graded
doses of compounds as in the preceding descriptions.
[0119] Assay for agonist. Evaluation of the molecules to elicit a
functional response in HEK-293 cells expressing MC4-R was
accomplished by measuring the accumulation of intracellular cAMP
following treatment. Confluent HEK-293 cells expressing MC4-R
receptors were detached by enzyme free cell suspension buffer.
Cells were suspended in Earle's Balanced Salt Solution containing
10 mM HEPES (pH 7.5), 1 mM MgCl.sub.2, 1 mM glutamine, 0.5% albumin
and 0.3 mM 3-isobutyl-1-methyl-xanthine (IBMX), a phosphodiesterase
inhibitor. The cells were plated in a 96 well plates at a density
of 0.5.times.10.sup.5 cells per well and pre-incubated for 30
minutes. Test compounds (dissolved in dimethylsulfoxide (DMSO) at a
concentration range of 0.05-5000 nM) were added in a total assay
volume of 200 .mu.L and cells were incubated for 1 hour at
37.degree. C. The concentration of DMSO was always held at 1% in
the assay mixture. NDP-.alpha.-MSH was used as the reference
agonist. At the end of the incubation period the cells were
disrupted by the addition of 50 .mu.L of lysis buffer from a cAMP
EIA kit (Amersham). Complete rupture of the cells was obtained by
pipetting the cells multiple times. cAMP levels in the cell lysates
were measured after appropriate dilution using the EIA kit
(Amersham) method. Data analysis and EC.sub.50 values were
determined by using nonlinear regression analysis with Prism
Graph-Pad software. Compounds at a concentration of 5000 nM that
had a response ratio compared to NDP-.alpha.-MSH of 0.7 (70%) and
above were classified as full agonists. Compounds with a ratio from
0.1 to 0.7 (10% to 70%) were classified as partial agonists.
Compounds with a response ratio of less than 0.1 (10%) were
evaluated for antagonistic activity.
[0120] Assay for Neutral Antagonist. Compounds with a high affinity
for binding to MC4-R membranes but with less efficacy
(EC.sub.50>1000 nM) and low response ratio (<0.1) were
analyzed for their ability to antagonize the stimulatory effect of
the agonist NDP-.alpha.-MSH. These studies were carried out in
HEK-293 cells expressing the MC4-R receptor. Cells were incubated
with the compounds in the presence of the agonist NDP-.alpha.-MSH
and the extent of antagonism was measured by the decrease in the
level of intracellular cAMP. Screening the compounds for
antagonists was done at a single concentration of NDP-.alpha.-MSH
(1.0 nM) over a compound concentration range of 0.5-5000 nM.
Studies were extended further for compounds exhibiting strong
antagonism to derive the pA.sub.2 value from Schild analysis.
[0121] Experimental details were similar to the analysis for
agonistic activity described above. Briefly, cells were
pre-incubated for 30 minutes with the test compounds at
concentrations between 0.5 nM and 5000 nM. The cells were then
stimulated with NDP-.alpha.-MSH at a concentration of 1 nM for 1
hour. For Schild analysis, the interactions were studied using at
least 3 concentrations of the compounds, separated by a log unit,
over a full range of the agonist (0.005-5000 nM). cAMP levels was
measured in the cell lysates after appropriate dilution. Nonlinear
regression analysis with Prism Graph-Pad software was used for
Schild analysis and to obtain EC.sub.50 values. pA.sub.2 values
were derived from the Schild plot of the data.
[0122] Cell culture and doxycycline induction. HEK-293 cells that
express hMC4-R using a doxycycline-regulated expression system were
grown in DMEM (Gibco) supplemented with 10% fetal bovine serum, 250
.mu.g/mL Zeocin (Invitrogen), 5 .mu.g/mL Blasticidin (Invitrogen)
and 2 mM glutamine (Invitrogen.). Cells were maintained at
37.degree. C. in an environment of 5% CO.sub.2. To induce receptor
expression, cells (at approximately 85% confluency) were exposed to
doxycycline for 18 hours.
[0123] Membrane preparation and radioligand binding. After
doxycycline induction, media containing doxycycline was removed and
cells were washed with cold PBS. To disrupt cells from culture
flasks, 10 mL of cold buffer (10 mM Tris, pH 7.4, 5 mM EDTA) was
added to culture flasks and allowed to incubate for 10 minutes at
4.degree. C. Dislodged cells were transferred to assay tubes and
homogenized for 15 seconds with a Polytron homogenizer. The cell
homogenates were centrifuged for 30 minutes at 20000 rpm. The
supernatant was removed and pellets were resuspended in storage
buffer (50 mM Tris, pH 7.4, 1 mM EDTA). Aliquots of 1 mL were made
from each resuspended pellet and frozen at -80.degree. C. A
Bradford protein assay was used to determine protein concentration
with BSA as the standard. On the day of an assay, membrane
homogenates were thawed and incubated (3 .mu.g protein/well) at
37.degree. C. for 90 minutes with [I.sup.125]-NDP-.alpha.-MSH at
concentrations from 0.02 nM to 1 nM in 200 .mu.L assay buffer (25
mM HEPES, pH 7.4, 100 mM NaCl, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2,
0.3 mM 1,10 phenanthroline/DMSO and 0.2% BSA). The reaction was
stopped by filtration over GF/C filters (presoaked in 0.5%
polyethylenimine) with five washes of cold PBS using a Brandel
harvester. Non-specific binding was determined with the addition of
1 .mu.M NDP-.alpha.MSH. Membrane filters were punched and counted
on a PerkinElmer Cobra gamma counter. B.sub.max and K.sub.D values
were determined by Scatchard analysis.
[0124] Agonist stimulation in cells using a doxycycline-regulated
expression system. After induction of MC4-R expression with
doxycycline, media containing doxycycline was removed and cells
were detached by incubation in enzyme-free cell suspension buffer.
Assay buffer consisting of Hank's Balanced Salt Solution, 10 mM
HEPES (pH 7.4), 5 mM MgCl.sub.2, 1 mM glutamine, 1 mg/mL BSA and
0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX) was added, cells were
transferred to assay tubes and centrifuged for 4 minutes at 1600
rpm. Following centrifugation, cells were re-suspended in the assay
buffer, transferred to 96 well plates at a density of 5 to
10.times.10.sup.4 cells per well and incubated for 10 minutes at
37.degree. C. and 5% CO.sub.2. Cells were exposed to test compounds
(10 .mu.M to 10 .mu.M) for 15 minutes at 37.degree. C. in an assay
volume of 200 .mu.L. At the end of the incubation period, cells
were lysed by the addition of 50 .mu.L of lysis buffer from a cAMP
EIA kit (Amersham) or frozen at -80.degree. C. cAMP levels in the
cell lysates were measured using the Adenylyl Cyclase Activation
FlashPlate Assay (Perkin Elmer). FlashPlates were incubated at room
temperature for 12 hours and activity determined by a Tri-Lux
Microbeta counter (Perkin Elmer). EC.sub.50 and E.sub.max values
for the dose-response curves were determined using PRISM (GraphPad
Software Inc., San Diego, Calif.).
[0125] Penile erection induction. The ability of compounds to
induce penile erection (PE) in male rats was evaluated with
selected compounds. Male Sprague-Dawley rats weighing 200-250 g
were kept on a 12 hour on/off light cycle with food and water ad
libitum. All behavioral studies were performed between 10 A.M. and
5 P.M. Groups of 4-8 rats were treated with compounds at a variety
of doses via intravenous (IV) or intracerebroventricular (ICV)
routes. Immediately after treatment, rats were placed into
individual polystyrene cages (27 cm long, 16 cm wide, and 25 cm
high) for behavioral observation. Rats were observed for 30 to 60
minutes following IV administration or 120 minutes following ICV
administration, and the number of yawns, grooming bouts and PEs
were recorded in 10-minute bins. Controls utilized carrier without
the test compound. Mean PEs in control groups were 0.17 to 0.5
PEs/rat by IV administration in various experiments and between 1-2
PEs/rat by ICV administration in various experiments, and thus only
PEs with statistically relevant increases over the mean PEs in
control groups are reported as inducing PEs. A PE response in IV
animals greater than the mean PEs in control groups but less than
1.0 PEs/rat, particularly with less than all animals responding,
was treated as equivocal, and not necessarily distinguishable from
vehicle control. In selected instances, compounds with equivocal
results in the IV model were tested for penile response in an ICV
model, and compounds without a statistically relevant increase over
the mean PEs in ICV control groups were determined to not induce
PEs.
[0126] ICV food intake and body weight change. Change in food
intake and body weight was evaluated for selected compounds. Rats
with indwelling intracerebroventricular cannulas (ICV rats) were
obtained from Hilltop Lab Animals, Inc. (Scottdale, Pa.). Animals
were individually housed in conventional plexiglass hanging cages
and maintained on a controlled 12 hour on/off light cycle. Water
and powdered (LabDiet, 5P00 Prolab RMH 3000) or pelleted (Harlan
Teklad 2018 18% Protein Rodent Diet) food was provided ad libitum.
For 1 week before treatment, 24-hour food intake and body weight
change was recorded to assess a baseline for the group during
vehicle treatment. The rats were dosed ICV with vehicle or selected
compounds (1-3 nmol). The changes in body weight and food intake
for the 24 hour period after dosing were determined. The changes in
body weight and food intake for the 48 hour and 72 hour periods
after dosing were also measured to determine reversal of changes in
body weight and food intake effects back to baseline levels.
[0127] IV and IP food intake and body weight change. Change in food
intake and body weight was evaluated for selected compounds. Male
Sprague-Dawley rats or male C57BL/6 mice were utilized. Animals
were individually housed in conventional plexiglass hanging cages
and maintained on a controlled 12 hour on/off light cycle. Water
and powdered (LabDiet, 5P00 Prolab RMH 3000) or pelleted (Harlan
Teklad 2018 18% Protein Rodent Diet) food was provided ad libitum.
For 1 week before treatment, 24-hour food intake and body weight
change was recorded to assess a baseline for the group during
vehicle treatment. The rats were dosed IV with vehicle or selected
compounds (0.5-3 mg/kg, and in some cases up to 10 mg/Kg) or dosed
intraperitoneally (IP) with vehicle or selected compounds (0.5-10
mg/kg, and in some cases up to 50 mg/kg). Mice were dosed IP. The
changes in body weight and food intake for the 24 hour period after
dosing were determined. The changes in body weight and food intake
for the 48 hour and 72 hour periods after dosing were also measured
to determined reversal of changes in body weight and food intake
effects back to baseline levels.
[0128] Determination of mass and nuclear magnetic resonance
analysis. The mass values were determined using a Waters MicroMass
ZQ device utilizing a positive mode. Mass determinations were
compared with calculated values and expressed in the form of mass
weight plus one (M+1 or M+H).
[0129] Proton NMR data was obtained using a Bruker 300 MHz
spectrometer. The spectra were obtained after dissolving compounds
in a deuteriated solvent such as chloroform, DMSO, or methanol as
appropriate.
[0130] Summary of cAMP Response in High and Low Receptor Density
Studies. Table 2 lists the compounds employed in low density and
high density receptor studies. Table 3 summarizes the data obtained
with those compounds.
TABLE-US-00002 TABLE 2 Compound or Example No. Molecular Structure
NDP-.alpha.-
Ac-Ser-Tyr-Ser-Met-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH.sub.2
MSH .alpha.-MSH
Ac-Ser-Tyr-Ser-Nle-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH.sub.2
(SEQ ID NO: 1) MT-II
Ac-Nle-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-NH.sub.2 PT-141 cyclo
2,7[Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys]-OH SHU-9119 cyclo
2,7[Ac-Nle-Asp-His-D-Nal 2-Arg-Trp-Lys]-NH.sub.2 AgRP (83- (AgRP
(83-132)) 132) 1
NH.sub.2--(CH.sub.2).sub.6C(.dbd.O)-Ser(Bzl)-D-Phe(4-Cl)-Arg-Trp-NH.sub.-
2 2 ##STR00001## 3 ##STR00002## 4 ##STR00003## 5 ##STR00004## 6
##STR00005## 7 ##STR00006## 8 ##STR00007## 9 ##STR00008## 10
##STR00009## 11 ##STR00010## 12 ##STR00011## 13
NH.sub.2--(CH.sub.2).sub.6--C(.dbd.O)-Ser(Bzl)-D-Phe(4-Cl)-Arg-Trp-Asp--
NH.sub.2 14
NH.sub.2--(CH.sub.2).sub.6--C(.dbd.O)-Ser(Bzl)-D-Phe(4-Cl)-Arg-Trp-Asp--
Phe-NH.sub.2 15
NH.sub.2--(CH.sub.2).sub.6--C(.dbd.O)-Ser(Bzl)-D-Phe(4-Cl)-Arg-Trp-Ala--
NH.sub.2 16 ##STR00012## 17 ##STR00013## 18 ##STR00014## 19
##STR00015## 20 ##STR00016## 21 ##STR00017## 22 ##STR00018## 23
heptanoyl-Thr(Bzl)-D-Phe(4-Cl)-Arg-Trp-NH.sub.2 24
2-naphthylacetyl-1-amino-1-cyclohexane-carbonyl-D-Phe(4-Cl)-Arg-Trp-NH.-
sub.2 25 ##STR00019## 26 ##STR00020## 27 ##STR00021## 28
##STR00022## 29 ##STR00023## 30 ##STR00024## 31 ##STR00025## 32
##STR00026## 33 ##STR00027## 34 ##STR00028## 35 ##STR00029## 36
##STR00030## 37 ##STR00031## 38 ##STR00032## 39 ##STR00033## 40
##STR00034##
TABLE-US-00003 TABLE 3 0.1 ng/mL 10 ng/mL Doxycycline Doxycycline
Efficacy/ Efficacy/ Penile Food Intake Compound Intrinsic EC.sub.50
Intrinsic EC.sub.50 Erection ICV Rat Mouse No. Activity (nM)
Activity (nM) ICV IV Rat IV/IP IP NDP-.alpha.- 1 1 1 0.5 .dwnarw.
MSH.sup.1 .alpha.-MSH 0.9 1 .dwnarw. MT-II.sup.2 0.77 0.5 >1 0.4
Yes** Yes** .dwnarw. PT-141 0.90 3 1 0.9 Yes** Yes** .dwnarw.
SHU-9119.sup.3 None None No No .uparw. AGRP (83-132).sup.3 None
None No No .uparw. 1 None 0.97 21 No .dwnarw. .dwnarw. 2 None 0.56
57 No 3 None 0.26 61 4 None None .dwnarw. 5 None 0.46 117 No 6 None
0.43 39 No 7 None 0.38 72 No 8 None None No 9 None 0.73 22 No No
.dwnarw. 10 0.23 38 0.69 26 Yes* .dwnarw. 11 None 0.49 61 .dwnarw.
12 None None .dwnarw. 13 None 0.72 34 No .dwnarw. .dwnarw. 14 None
0.64 141 No .dwnarw. .dwnarw. 15 None 0.67 55 No .dwnarw. .dwnarw.
16 None 0.63 180 No .dwnarw. 17 None 0.67 847 No No .dwnarw.
.dwnarw. 18 None 0.29 281 .dwnarw. 19 None 0.22 173 No .dwnarw.
.dwnarw. 20 None 0.38 215 No .dwnarw. 21 None None No .dwnarw. 22
None 0.30 64 No .dwnarw. 23 None 0.75 184 No .dwnarw. 24 None None
.dwnarw. 25 None None .dwnarw. 26 None 0.78 31 No .dwnarw. .dwnarw.
27 None 0.61 129 No No .dwnarw. .dwnarw. 28 0.29 106 0.75 24 No
.dwnarw. .dwnarw. 29 0.25 45 0.77 34 Yes* .dwnarw. 30 None 0.18 51
No .dwnarw. 31 None 0.27 39 No .dwnarw. 32 None None No No .dwnarw.
33 None 0.73 50 No .dwnarw. 34 None None No .uparw. 35 None None No
.uparw. 36 None 0.98 4 37 0.69 2.1 0.88 0.6 38 None 1.0 10.0 39
0.77 10 1.0 1.1 40 1.0 3.1 1.0 0.5 Yes *Weak response observed
without all animals in the group responding. **Robust response with
all animals in the group responding and some exhibiting multiple
PEs. .sup.1NDP-.alpha.-MSH caused 22% decrease in ICV (1 nmole)
food intake in 24 hrs. .sup.2MT-II caused 54% decrease in ICV (1
nmole) food intake in 24 hrs. .sup.3Functional antagonists known
not to cause PE response.
[0131] Applicants have surprisingly and unexpectedly found that
melanocortin receptor-specific compounds, which may be either
peptide-based or small molecules, may be selected by the methods of
this invention by determining intrinsic activity of the compound in
both low and high density melanocortin receptor systems. In one
aspect, compounds are selected which are useful for attenuating
food intake and body weight gain generally such as for treatment of
obesity and related energy homeostasis diseases, conditions and
syndromes, which compounds do not induce a substantial, or any,
sexual response in a mammal, such as an erectile response in a
male. It has been recognized for several years that MC4-R agonists
may be employed for the treatment of obesity and related energy
homeostasis diseases, conditions and syndromes. However, a number
of melanocortin-receptor specific compounds and agents heretofore
evaluated for use in treatment of obesity and related conditions
have had unacceptable side effects relating to initiation or
induction of a sexual response, including a penile erection
response in males. In part, compounds disclosed herein, and
compounds selected by methods of the invention, do not generally
induce any sexual response, and/or do not induce a substantial
sexual response.
[0132] It has surprisingly and unexpectedly been discovered that
compounds, which may be peptides, peptide-derived molecules, or
small molecules, including small molecules with a ring core
structure, which compounds have ascertainable intrinsic activity in
a low density melanocortin receptor system, and specifically a low
hMC4-R density system, will induce a sexual response in a mammal,
such as an erectile response in a male, while compounds that do not
have ascertainable intrinsic activity in a low density melanocortin
receptor system, and specifically a low hMC4-R density system, will
generally not induce a sexual response in a mammal, such as an
erectile response in a male.
[0133] A first embodiment of the present invention provides a
method for discriminating melanocortin receptor-specific compounds,
and specifically MC4-R specific compounds, based upon an in vitro
assay system, between those compounds likely to induce a sexual
response and those likely not to induce a sexual response.
Compounds that do not have an ascertainable intrinsic activity in a
system with a low density of melanocortin receptors, and
specifically a low density hMC4-R system, but which do have an
ascertainable intrinsic activity in a high density melanocortin
receptor system, and specifically a high hMC4-R density system, may
be selected for use in attenuating food intake and body weight gain
generally such as for use in the treatment of obesity, without
inducing a substantial sexual response or any sexual response. The
method of the first embodiment thus includes providing a low
density melanocortin receptor system, and preferably a low density
hMC4-R system, providing a high density melanocortin receptor
system, preferably a high density hMC4-R system, testing a compound
in each system, and selecting a compound for use in attenuating
food intake and body weight gain generally such as for treatment of
obesity without inducing a substantial or any sexual response by
selecting a compound with no ascertainable intrinsic activity in
the low density melanocortin receptor system but with ascertainable
intrinsic activity in the high density melanocortin receptor
system.
[0134] Compounds with an ascertainable intrinsic activity in the
high density hMC4-R system have an intrinsic activity of greater
than about 0.1 (10%), such as based on maximal stimulation of
adenylyl cyclase achievable by the compound in the same high
density hMC4-R system where the maximal stimulation achieved by
.alpha.-MSH or NDP-.alpha.-MSH is designated as an intrinsic
activity of 1.0 (100%). Preferably, such compounds have an
intrinsic activity of greater than about 0.2 (20%), more preferably
greater than about 0.3 (30%), still more preferably greater than
about 0.5 (50%), and most preferably greater than about 0.7 (70%).
Compounds that do not have an ascertainable intrinsic activity in a
low density melanocortin receptor system, and specifically a low
density hMC4-R system, have either no measurable intrinsic
activity, or alternatively if the compounds have a measurable
intrinsic activity, have an intrinsic activity of less than about
0.1 (10%), such as based on maximal stimulation of adenylyl cyclase
achievable by the compound in the same low density hMC4-R system
where the maximal stimulation achieved by .alpha.-MSH or
NDP-.alpha.-MSH is designated as an intrinsic activity of 1.0
(100%).
[0135] The method disclosed above in the first embodiment may be
used in combination with the methods disclosed in a related
application entitled Compounds and Methods for Treatment of
Obesity, U.S. patent application Ser. No. ______, filed
concurrently herewith, Attorney Docket No. 056291-5365, and the
specification thereof is incorporated herein by reference. The
Compounds and Methods for Treatment of Obesity application
discloses a method for selecting melanocortin receptor-specific
compounds for treatment of obesity and related disorders, and in
particular compounds for treatment of obesity and related energy
homeostasis or feeding disorders characterized by excess weight
gain, but which compounds do not induce a sexual response,
including a penile response. In one embodiment of the methods of
the Compounds and Methods for Treatment of Obesity application, a
compound, which may be a peptide, peptide-derivative, or a small
molecule, is selected which is an agonist or partial agonist as to
MC4-R in a normal density melanocortin receptor system, but which
may further be characterized in that the compound has high
affinity, and on approximately the same order, as to MC4-R with
respect to both .alpha.-MSH and AgRP, and thus attenuates the
binding of both .alpha.-MSH and AgRP to MC4-R. This compound may
further optionally be characterized in that it has a Ki (nM) at
MC4-R, determined with respect to NDP-.alpha.-MSH, that is half or
less than half, and preferably substantially less than half, of the
EC.sub.50 (nM) at MC4-R. Thus the EC.sub.50 (nM) at MC4-R may be 2
times, 5 times, 10 times, 20 times, or more higher than the
corresponding Ki (nM) at MC4-R, determined with respect to
NDP-.alpha.-MSH. Thus, a compound may be selected on the basis of
intrinsic activity in both a low and high density melanocortin
receptor system, and may further, optionally and additionally be
selected based upon one or more methods disclosed in the Compounds
and Methods for Treatment of Obesity application, including the
foregoing.
[0136] A second embodiment of the present invention provides a
method for discriminating melanocortin receptor-specific compounds,
and specifically MC4-R specific compounds, based upon an in vitro
assay system, between those compounds likely to induce a sexual
response and those likely not to induce a sexual response.
Compounds that have an ascertainable intrinsic activity in a low
density melanocortin receptor system, and specifically a low
density hMC4-R system, and which further have an ascertainable
intrinsic activity in a high density melanocortin receptor system,
and specifically a high density hMC4-R system, may be selected for
use in the treatment of sexual dysfunction, such as male erectile
dysfunction and female sexual dysfunction. The method of the second
embodiment thus includes providing a low density melanocortin
receptor system, and preferably a low density hMC4-R system,
providing a high density melanocortin receptor system, and
preferably a high density hMC4-R system, testing a compound in each
system, and selecting a compound for treatment of sexual
dysfunction by selecting a compound with ascertainable intrinsic
activity in the low density melanocortin receptor system and
ascertainable intrinsic activity in the high density melanocortin
receptor system.
[0137] Compounds with an ascertainable intrinsic activity in the
low density hMC4-R system are characterized, in one aspect, by
having an intrinsic activity of greater than about 0.1 (10%), based
on maximal stimulation of adenylyl cyclase achievable by the
compound in the same low density hMC4-R system where the maximal
stimulation achieved by .alpha.-MSH or NDP-.alpha.-MSH is
designated as an intrinsic activity of 1.0 (100%). Preferably such
compounds have an intrinsic activity of greater than about 0.2
(20%), more preferably greater than about 0.3 (30%), still more
preferably greater than about 0.5 (50%), and most preferably
greater than about 0.7 (70%). Compounds with an ascertainable
intrinsic activity in the high density hMC4-R system have an
intrinsic activity of greater than about 0.1 (10%), based on
maximal stimulation of adenylyl cyclase achievable by the compound
in the same high density hMC4-R system where the maximal
stimulation achieved by .alpha.-MSH or NDP-.alpha.-MSH is
designated as an intrinsic activity of 1.0 (100%). Preferably such
compounds have an intrinsic activity of greater than about 0.2
(20%), more preferably greater than about 0.3 (30%), still more
preferably greater than about 0.5 (50%), and most preferably
greater than about 0.7 (70%).
[0138] Preferably, such compounds selected for use in treatment of
sexual dysfunction by the second embodiment described above can
further be characterized in that such compounds are agonists as to
MC4-R, which is to say, have an intrinsic activity of greater than
about 0.7 (70%), such as based on maximal stimulation of adenylyl
cyclase achievable by the compound in a normal density hMC4-R
system where the maximal stimulation achieved by .alpha.-MSH or
NDP-.alpha.-MSH is designated as an intrinsic activity of 1.0
(100%).
[0139] Compounds tested by the methods of the second embodiment are
not likely to be effective for use in the treatment of sexual
dysfunction, even if such compounds are agonists as to MC4-R as
determined in a normal density hMC4-R system, if such compounds are
inactive or have a low intrinsic activity, such as below about 0.1
(10%), preferably below about 0.3 (30%), based on maximal
stimulation of adenylyl cyclase achievable by the compound in a
normal density hMC4-R system where the maximal stimulation achieved
by .alpha.-MSH or NDP-.alpha.-MSH is designated as an intrinsic
activity of 1.0 (100%).
[0140] A third embodiment of the present invention provides a
method for selecting neutral antagonist compounds that may be
employed for treatment of energy homeostasis or feeding disorders
or conditions and/or for attenuating food intake and weight gain
generally. Such energy or feeding disorders and conditions include,
but are not limited to, body weight disorders such as cachexia,
sarcopenia and wasting syndrome or disease, and alternatively or
additionally treatment of inflammation and immune disorders. Such
energy or feeding disorders and conditions may further include
treatment of obesity and related energy homeostasis or feeding
disorders characterized by excess weight gain. Such compounds are
further characterized in that they at least substantially do not
induce and/or do not induce at all a sexual response in a mammal.
Applicants have surprisingly and unexpectedly found that certain
compounds which are classified as antagonists with respect to MC4-R
in a normal density hMC4-R system, and thus by definition are not
agonists or partial agonists as to as to MC4-R, are useful for
attenuating food intake and weight gain generally such as for
treatment of obesity and related energy homeostasis diseases,
conditions and syndromes. Thus, the compounds of Examples 4, 12,
13, 23 and 26 of the Compounds and Methods for Treatment of Obesity
application are classified as antagonists, with pA.sub.2 values as
given therein. The compounds of Examples 4, 12, 13, 23 and 26 of
the Compounds and Methods for Treatment of Obesity application each
caused a decrease in body weight under the described experimental
conditions. Such neutral antagonist compounds may be selected as at
least substantially not inducing a sexual response in a mammal,
including at least substantially not inducing a penile erection in
a male, if such compounds are inactive in both a low and high
density melanocortin receptor system. Thus, such compounds have no
ascertainable intrinsic activity in both a low and high density
melanocortin receptor system, and specifically a low and high
density hMC4-R system, where in each system the maximum measurable
intrinsic activity, if any is less than about 0.1 (10%), based on
maximal stimulation of adenylyl cyclase achievable by the compound
in the same low or high density hMC4-R system where the maximal
stimulation achieved by .alpha.-MSH or NDP-.alpha.-MSH is
designated as an intrinsic activity of 1.0 (100%).
[0141] It may thus be seen that the invention, as described in the
foregoing embodiments and elsewhere in this specification, provides
new, novel and useful methods for selecting melanocortin
receptor-specific compounds for modulating food intake and weight
gain generally, such as for treating obesity and related energy
homeostasis diseases, conditions and syndromes. Thus, the methods
of the invention may be used to screen and select compounds with
the desired pharmacological profiles. Heretofore, as described in
the Background of the Invention, methods employed by others have
simply provided for screening for agonist status, typically as to
MC4-R, selecting agonist compounds, and testing such compounds.
This invention illustrates that significantly different mechanisms
of action are implicated in selection of compounds for the specific
indication of obesity and related energy homeostasis diseases,
conditions and syndromes, and further selection of compounds which
do not cause unintended effects, such as inducing a sexual
response.
[0142] The invention, in part, relates to the discovery that
determining the efficacy of specific compounds in terms of cAMP
expression in a low density melanocortin receptor system, and
specifically a low density hMC4-R system, and in terms of a high
density melanocortin receptor system, and specifically a high
density hMC4-R system, provides valuable and specific information
relating directly to the potential clinical utility of such
compounds. This thus permits compounds to readily be identified and
categorized as compounds with potential utility for attenuating
food intake and weight gain generally such as for treatment of
obesity and related energy homeostasis diseases, at least
substantially without causing sexual responses, and conversely
permits compounds to readily be identified and categorized as
compounds for treatment of sexual dysfunction, including male
erectile dysfunction and female sexual disorder.
[0143] It may be further be seen that the invention provides
methods that may readily be adapted for use to screen, select and
characterize any melanocortin receptor-specific compound, whether a
peptide, peptide derivative, small molecule or otherwise. It may
further be seen that the methods may be adapted, by selecting
different reactants, reaction conditions, reagents, or the like,
and still remain within the scope of this invention. Thus, by way
of example, and not limitation, other reference compounds may be
used in place of .alpha.-MSH, such as other known or hereafter
development melanocortin agonists, other variable gene expression
regulatory systems may be employed, other vectors and genetic
constructs may be employed, other cell lines may be employed, and
the like. In particular, other means and methods for determining
the functional activity of a compound, including but not limited to
the means and methods heretofore described, may be employed. In
general, once the principles of the invention are understood, one
of ordinary skill in the art can determine and develop alternative
means and methods of practicing the invention. It is intended to
include all such variations within the invention.
[0144] While the invention is exemplified in terms of
differentiation of the effects of melanocortin receptor-specific
agents between effects associated with attenuation of food intake
and/or body weight and those effects associated with a sexual
response, including a penile erection response in males, it may
readily be seen that the methods of the invention may be applied to
other effects and responses associated with or resulting from
administration of a melanocortin receptor-specific agent. In
particular, systemic effects resulting from administration of a
melanocortin receptor-specific agent, particularly an agonist
agent, such as pressor effects resulting in an increase in blood
pressure, pica behavior, disruption of normal behavioral satiety
sequences and various other systemic effects, are believed to be
associated with potent agonist responses derived through central
melanocortin agents, and are thus believed to be associated with
the mechanisms resulting in a sexual response, and not with the
mechanisms associated with attenuation of food intake and/or body
weight. Thus in another aspect the invention provides a method for
differentiation and selection of melanocortin receptor-specific
agents useful for attenuating food intake but which agents do not
have, or do not substantially have, a pressor effect or other
effects associated with potent agonist responses or, in particular,
agonist responses or intrinsic activity in a low density
melanocortin receptor system, and in particular a low density MC4-R
system.
[0145] In each of the following examples, the data presented in
Table 3 is incorporated by reference, including specifically the
data on "Efficacy/Intrinsic Activity" and "EC.sub.50 (nM)" in both
the low density hMCR-4 density system (0.1 ng/ML doxycycline) and
the high density hMCR-4 density system (10 ng/ML doxycycline). The
invention is illustrated by the following non-limiting examples, it
being understood that similar or related methods may be employed
with different compounds, and yet be within the scope of the
invention.
Example 1
NH.sub.2--(CH.sub.2).sub.6--C(.dbd.O)-Ser(Bzl)-(D)Phe(4-Cl)-Arg-Trp-NH.sub-
.2
[0146] The peptide compound
NH.sub.2--(CH.sub.2).sub.6--C(.dbd.O)-Ser(Bzl)-(D)Phe(4-Cl)-Arg-Trp-NH.su-
b.2 was synthesized by peptide synthesis methods as disclosed in
International Patent Application No. PCT/US02/22196. The molecular
weight was determined to be 845. Competitive inhibition testing of
the compound yielded the following results:
TABLE-US-00004 MC1-R MC3-R MC4-R MC5-R Inhibition at 1 .mu.M
(NDP-.alpha.-MSH) In Normal System 33% 94% 99% 73% Ki (nM)
(NDP-.alpha.-MSH) In Normal System 351 27 3 208 Low Receptor
Density System Efficacy/Intrinsic High Receptor Density System
Activity EC.sub.50 (nM) Efficacy/Intrinsic Activity EC.sub.50 (nM)
None N/A 0.97 21
[0147] In a cAMP assay using MC1-R, MC4-R and MC5-R, at 1 .mu.M
concentrations the compound of Example 1 was a partial agonist at
MC4-R.
[0148] The EC.sub.50 (nM) at MC4-R in a normal system was
determined to be 296 with intrinsic activity of 0.32.
[0149] In rat model IV feeding studies at 0.75 mg/kg dose levels, a
maximal 37% decrease was observed in food intake for a period of 24
hours. In rat model ICV feeding studies at 1 nmol dose levels, a
maximal 21% decrease was observed in food intake for a period of 24
hours. In mouse model IP feeding studies at 3 mg/kg dose levels, a
maximal 26% decrease was observed in food intake for a period of 4
hours. In mouse model IN feeding studies at 10 .mu.g/kg dose
levels, a maximal 16% decrease was observed in food intake for a
period of 4 hours.
[0150] In rat model penile behavior induction experiments by ICV
administration of doses from 0.01 to 5 nmol, no penile erection
response was observed.
Example 2
N-(3-{(S)-1-[(R)-2-Amino-3-(2,4-dichloro-phenyl)-propionyl]-4-[2-(1H-indol-
-3-yl)-ethyl]-3-oxo-piperazin-2-yl}-propyl)-guanidine
[0151] A compound of the following structure:
##STR00035##
was synthesized by methods described in U.S. patent application
Ser. No. 10/762,079. The molecular weight was determined to be
557.5 ESI-MS (M+1). Competitive inhibition testing of the compound
yielded the following results (average of triplicates with actual
mean values described):
TABLE-US-00005 MC1-R MC3-R MC4-R MC5-R Inhibition at 1 .mu.M
(NDP-.alpha.-MSH) In Normal System 7% 57% 96% 35% Ki (nM)
(NDP-.alpha.-MSH) In Normal System 1409 533 15 1578 Low Receptor
Density System Efficacy/Intrinsic High Receptor Density System
Activity EC.sub.50 (nM) Efficacy/Intrinsic Activity EC.sub.50 (nM)
None N/A 0.56 57
[0152] In a cAMP assay using MC1-R, MC4-R and MC5-R, at 1 .mu.M
concentrations the compound of Example 2 was a partial agonist at
MC4-R, MC5-R and MC1-R.
[0153] The EC.sub.50 (nM) at MC4-R in a normal system was
determined to be 604 with intrinsic activity of 0.3.
[0154] In rat model penile behavior induction experiments at 0.3 to
3 .mu.g/kg (IV), the compound did not cause penile erection
behavior.
Example 3
N-{3-[1-[2(R)-Amino-3-(4-chloro-2-fluoro-phenyl)-propionyl]-5(S)-methyl-4--
(2-naphthalen-2-yl-ethyl)-piperazin-2(S)-yl]-propyl}-guanidine
[0155] The following compound was synthesized by methods described
in U.S. patent application Ser. No. 10/837,519, using
2-naphthylacetic acid as J-COOH, D-alanine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3, Fmoc-Arg(Boc).sub.2-OH as
Prt-NH--CH(R.sub.2)--COOH, and Boc-D-2-fluoro,4-chloro-Phe-OH as
Q-COOH. It was tested as described above with the results shown.
The mass was analyzed as 553 (M+H).
TABLE-US-00006 ##STR00036## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 57% 82% 99% 59% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 109 186
6 204 Low Receptor High Receptor Density System Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A 0.26 61
[0156] In a cAMP assay using MC1-R, MC4-R and MC5-R, at 1 .mu.M
concentrations the compound of Example 3 exhibited no intrinsic
activity (inactive) at MC4-R and MC5-R and was a partial agonist at
MC1-R.
Example 4
N-{3-[1-[2(R)-Amino-3-(2,4-dichloro-phenyl)-propionyl]-5(R)-isobutyl-4-(2--
naphthalen-2-yl-ethyl)-piperazin-2(S)-yl]-propyl}-guanidine
[0157] The following compound was synthesized by the methods of
both Schemes 3 and 5 described in U.S. patent application Ser. No.
10/837,519, using 2-naphthylacetic acid as J-COOH, D-Leucinol as
NH.sub.2--CH(R.sub.5)--CH(R.sub.4)--OH, D-leucine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3, Fmoc-L-Arg(Boc).sub.2-OH as
Prt-NH--CH(R.sub.2)--COOH, and Boc-D-2,4-dichloro-Phe-OH as Q-COOH.
It was tested as described above with the results shown. The mass
was analyzed as 611.1 (M+H).
TABLE-US-00007 ##STR00037## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 21% 64% 99% 75% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 1364 87
5 160 Low Receptor High Receptor Density System Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A None N/A
[0158] In a cAMP assay using MC1-R, MC4-R and MC5-R, at 1 .mu.M
concentrations the compound of Example 4 exhibited no intrinsic
activity at MC1-R and MC4-R, and was a partial agonist at MC5-R.
The compounds tested as antagonist as to MC4-R in a normal system
with a pA.sub.2 value of 7.7.
[0159] In mouse model IP feeding studies at 10 mg/kg dose levels, a
maximal 60% decrease was observed in food intake for a period of 4
hours.
Example 5
N-{3-[1-(2(R)-Amino-3-naphthalen-2-yl-propionyl)-5(R)-methyl-4-(2-p-tolyl--
ethyl)-piperazin-2(S)-yl]-propyl}-guanidine
[0160] The following compound was synthesized by the method of
Scheme 6 in U.S. patent application Ser. No. 10/837,519 using
4-methylphenylacetaldhyde as J-aldehyde, D-alanine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3, and Boc-D-2-Nal-OH as Q-COOH.
It was tested as described above with the results shown. The mass
was analyzed as 515.4 (M+H).
TABLE-US-00008 ##STR00038## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 32% 61% 95% 65% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 135 164
29 448 Low Receptor High Receptor Density System Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A 0.46 117
[0161] In a cAMP assay using MC1-R, MC3-R, MC4-R and MC5-R, at 1
.mu.M concentrations the compound of Example 5 exhibited no
intrinsic activity at MC3-R, and was a partial agonist at MC1-R,
MC4-R and MC5-R. An EC.sub.50 value of 370 nM on MC-4R in a normal
system was determined with an intrinsic activity of 0.6.
[0162] In rat model IV penile erection induction experiments at
doses ranging from 0.3 to 30 .mu.g/kg no penile erection response
was observed.
Example 6
N-{3-[1-(2(R)-Amino-3-naphthalen-2-yl-propionyl)-4-(2-1H-indol-3-yl-acetyl-
)-5(R)-methyl-piperazin-2(S)-yl]-propyl}-guanidine
[0163] The following compound was synthesized by the method of
Scheme 6 in U.S. patent application Ser. No. 10/837,519 using
indole-3-acetic acid as J-COOH, D-alanine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3, and Boc-D-2-Nal-OH as Q-COOH.
It was tested as described above with the results shown. The mass
was analyzed as 554.4 (M+H).
TABLE-US-00009 ##STR00039## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 20% 35% 90% 6% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 315 1822
35 1527 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A 0.43 39
[0164] In a cAMP assay using MC1-R, MC3-R, MC4-R and MC5-R, at 1
.mu.M concentrations the compound of Example 6 exhibited no
intrinsic activity at MC1-R and MC3-R, and was a partial agonist at
MC4-R and MC5-R. The EC.sub.50 value for MC4-R in a normal system
was over 1000 nM with an intrinsic activity of 0.2.
[0165] In rat model IV penile erection induction experiments at
doses ranging from 0.3 to 30 .mu.g/kg no penile erection response
was observed.
Example 7
N-{3-[1-(2(R)-Amino-3-naphthalen-2-yl-propionyl)-4-(2-1H-indol-3-yl-butyry-
l)-5(R)-methyl-piperazin-2(S)-yl]-propyl}-guanidine
[0166] The following compound was synthesized by the method of
Scheme 6 in U.S. patent application Ser. No. 10/837,519 using
indole-3-butyric acid as J-COOH, D-alanine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3, and Boc-D-2-Nal-OH as Q-COOH.
It was tested as described above with the results shown. The mass
was analyzed as 582.6 (M+H).
TABLE-US-00010 ##STR00040## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 0% 32% 88% 62% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 1203 657
90 271 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A 0.38 72
[0167] In a cAMP assay using MC1-R, MC3-R, MC4-R and MC5-R, at 1
.mu.M concentrations the compound of Example 7 exhibited no
intrinsic activity at MC1-R, MC3-R and MC5-R, and was a partial
agonist at MC4-R. The EC.sub.50 value for MC4-R in a normal system
was over 3000 nM with an intrinsic activity of 0.4.
[0168] In rat model penile behavior induction experiments by ICV
administration of doses from 0.01 to 10 nmol, no penile erection
response was observed.
Example 8
N-(3-{1-(2(R)-Amino-3-naphthalen-2-yl-propionyl)-5(R)-methyl-4-[2-(2-methy-
l-1H-indol-3-yl)-ethyl]-piperazin-2(S)-yl}-propyl)-guanidine
[0169] The following compound was synthesized by the method of
Scheme 6 in U.S. patent application Ser. No. 10/837,519 using
2-methyl-indole-3-acetaldehyde as J-aldehyde, D-alanine methyl
ester as NH.sub.2--CH(R.sub.5)--COOCH.sub.3, and Boc-D-2-Nal-OH as
Q-COOH. It was tested as described above with the results shown.
The mass was analyzed as 554.5 (M+H).
TABLE-US-00011 ##STR00041## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 73% 57% 95% 76% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 148 451
26 293 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A None N/A
[0170] In a cAMP assay using MC1-R, MC3-R, MC4-R and MC5-R, at 1
.mu.M concentrations the compound of Example 8 exhibited no
intrinsic activity at MC3-R, was a partial agonist at MC1-R, and
was an agonist at MC4-R and MC5-R. The EC.sub.50 value for MC4-R in
a normal system was over 384 nM with an intrinsic activity of
0.9.
[0171] In rat model penile behavior induction experiments by ICV
administration of doses from 0.01 to 10 nM, no penile erection
response was observed.
Example 9
N-(3-{1-(2(R)-Amino-(4-chloro-2-methyl-phenyl)-propionyl)-5(R)-methyl-4-[2-
-(1H-indol-3-yl)-ethyl]-piperazin-2(S)-yl}-propyl)-guanidine
[0172] The following compound was synthesized by the method of
Scheme 5 in U.S. patent application Ser. No. 10/837,519 using
indole-3-acetic acid as J-COOH, D-alanine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3, and
Boc-D-4-chloro-2-methyl-Phe-OH as Q-COOH. It was tested as
described above with the results shown. The mass was analyzed as
538.6 (M+H).
TABLE-US-00012 ##STR00042## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 40% 74% 98% 75% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 1730 177
5 360 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A 0.73 22
[0173] In a cAMP assay using MC1-R, MC3-R, MC4-R and MC5-R, at 1
.mu.M concentrations the compound of Example 9 exhibited no
intrinsic activity at MC1-R, MC3-R and MC5-R, and was a full
agonist at MC4-R. The EC.sub.50 value for MC4-R in a normal system
was over 7 nM with an intrinsic activity of 1.
[0174] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour decrease in food intake of 16%, and decrease in body weight of
2%, was observed.
[0175] In mouse model IP feeding studies at 3 mg/kg dose levels, a
maximal 10% decrease was observed in food intake for a period of 4
hours.
[0176] In rat model IV and ICV penile erection induction
experiments at doses ranging from 0.3 to 30 .mu.g/kg given IV and
at 0.01 to 10 nmole given ICV, no penile erection response was
observed.
Example 10
N-{3-[1-[2(R)-Amino-3-(4-chloro-2-dimethyl-phenyl)-propionyl]-5(R)-methyl(-
2-naphthalen-2-yl-acetyl)-piperazin-2(S)-yl]-propyl}-guanidine
[0177] The following compound was synthesized by the method of
Scheme 7 in U.S. patent application Ser. No. 10/837,519 using
2-naphthyl acetic acid as J-COOH, L-Orn(Boc) methyl ester as
NH.sub.2--CH(R.sub.2)--COOCH.sub.3, and
Boc-D-4-chloro-2-methyl-Phe-OH as Q-COOH. It was tested as
described above with the results shown. The mass was analyzed as
563.4 (M+H).
TABLE-US-00013 ##STR00043## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 3% 34% 96% 36% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 462 398
3 774 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) 0.23 38 0.69 26
[0178] In a cAMP assay for determination of agonist/antagonist
status, it was determined that the compound was a partial agonist
as to MC1-R and MC3-R, and an agonist as to MC4-R and MC5-R. The
EC.sub.50 value for MC4-R in a normal system was 18 nM with an
intrinsic activity of 0.9.
[0179] In rat model IV feeding studies at 10 mg/kg dose levels, a
maximal 33% decrease was observed in food intake for a period of 24
hours.
[0180] In mouse model IP feeding studies at 10 mg/kg dose levels, a
maximal 27% decrease was observed in food intake for a period of 4
hours.
[0181] In rat model IV penile erection induction experiments at
doses ranging from 0.3 to 30 .mu.g/kg, 0.5 to 0.7 mean penile
erections per rat were observed with not more than 66% animals
responding.
Example 11
N-{3-[1-[2(R)-Amino-3-(2,4-dichloro-phenyl)-propionyl]-5(R)-methyl-4-(2-na-
phthalen-2-yl-ethyl)-piperazin-2(S)-yl]-propyl}-guanidine
[0182] The following compound was synthesized by the methods of
both Schemes 3 and 5 in U.S. patent application Ser. No. 10/837,519
using 2-naphthylacetic acid as J-COOH, (R)-(-)-2-amino-1-propanol
as NH.sub.2--CH(R.sub.5)--CH(R.sub.4)--OH, Fmoc-L-Arg(Boc).sub.2-OH
as Prt-NH--C(R.sub.2)--COOH, D-alanine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3 and Boc-D-2,4-dichloro-Phe-OH as
Q-COOH. It was tested as described above with the results shown.
The mass was analyzed as 569.3 (M+H).
TABLE-US-00014 ##STR00044## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 20% 72% 99% 65% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 1134 95
2 362 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A 0.49 61
[0183] In a cAMP assay for determination of agonist/antagonist
status, it was determined that the compound was an antagonist as to
MC4-R with a pA.sub.2 value in a normal system of 7.8. At a 1 .mu.M
concentration it was inactive at MC1-R, MC3-R and MC5-R.
[0184] In rat model IV and IP feeding studies at 10 mg/kg dose
levels, a maximal 87% (IV) and 19% (IP) decrease was observed in
food intake for a period of 24 hours.
[0185] In mouse model IP feeding studies at 3 and 10 mg/kg dose
levels, a maximal 10% and 30% decrease, respectively was observed
in food intake for a period of 4 hours.
Example 12
N-{2-[2(S)-(3-Guanidino-propyl)-5(R)-isobutyl-4-(2-naphthalen-2-yl-ethyl)--
piperazin-1-yl]-1(R)-naphthalen-2-ylmethyl-2-oxo-ethyl}-acetamide
[0186] The following compound was synthesized by the method of
Scheme 5 in U.S. patent application Ser. No. 10/837,519 using
2-naphthylacetic acid as J-COOH, D-leucinol as
NH.sub.2--CH(R.sub.5)--CH(R.sub.4)--OH, D-leucine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3, and Boc-D-2-Nal-OH as Q-COOH as
described in Example 35 thereof. An acetyl group was attached to
the amino group of D-2-Nal by reaction of the compound of Example
35 with Ac-OSu in DMF. It was tested as described above with the
results shown. The mass was analyzed as 635.9 (M+H).
TABLE-US-00015 ##STR00045## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 0% 40% 100% 64% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 4616 474
6 378 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A None N/A
[0187] In a cAMP assay using MC1-R, MC3-R, MC4-R and MC5-R, at 1
.mu.M concentrations the compound of Example 13 exhibited no
intrinsic activity at MC1-R, MC3-R and MC4-R, and was a partial
agonist at MC5-R. At MC4-R in a normal system it tested as an
antagonist with a pA.sub.2 value of 7.3.
[0188] In mice model IP feeding studies at 10 mg/kg dose levels, a
maximal 46% decrease was observed in food intake for a period of 4
hours.
Example 13
NH.sub.2--(CH.sub.2).sub.6--C(.dbd.O)-Ser(Bzl)-D-Phe(4-Cl)-Arg-Trp-Asp-NH.-
sub.2
[0189] The peptide compound
NH.sub.2--(CH.sub.2).sub.6--C(.dbd.O)-Ser(Bzl)-D-Phe(4-Cl)-Arg-Trp-Asp-NH-
.sub.2 was synthesized by standard peptide synthesis methods as
described in International Patent Application No. PCT/US02/22196.
It was tested as described above with the results shown.
TABLE-US-00016 MC1-R MC3-R MC4-R MC5-R Inhibition at 1 .mu.M
(NDP-.alpha.-MSH) In Normal System 35% 73% 98% 48% Ki (nM)
(NDP-.alpha.-MSH) In Normal System 760 180 9 596 Low Receptor
Density System Efficacy/Intrinsic High Receptor Density System
Activity EC.sub.50 (nM) Efficacy/Intrinsic Activity EC.sub.50 (nM)
None N/A 0.72 34
[0190] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound in a normal system had
an EC.sub.50 (nM) of 568, showed intrinsic activity of 0.5, and was
a partial agonist as to MC4-R. It was inactive at MC1-R and MC5-R
and a partial agonist at MC3-R.
[0191] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour decrease in food intake of 25.7%, and decrease in body weight
of 2.1%, was observed. In IV feeding studies, a 24 hour decrease in
food intake of 6%, and decrease in body weight of 1%, was
observed.
[0192] In rat model IV penile erection induction experiments at 1
.mu.PK, no penile erections were observed.
Example 14
NH.sub.2--(CH.sub.2).sub.6--C(.dbd.O)-Ser(Bzl)-D-Phe(4-Cl)-Arg-Trp-Asp-Phe-
-NH.sub.2
[0193] The peptide corn pound
NH.sub.2--(CH.sub.2).sub.6--C(.dbd.O)-Ser(Bzl)-D-Phe(4-Cl)-Arg-Trp-Asp-Ph-
e-NH.sub.2 was synthesized by standard peptide synthesis methods as
described in International Patent Application No. PCT/US02/22196.
It was tested as described above with the results shown.
TABLE-US-00017 MC1-R MC3-R MC4-R MC5-R Inhibition at 1 Mm
(NDP-.alpha.-MSH) In Normal System 38% 66% 99% 52% Ki (nM)
(NDP-.alpha.-MSH) In Normal System 242 76 5 405 Low Receptor
Density System Efficacy/Intrinsic High Receptor Density System
Activity EC.sub.50 (nM) Efficacy/Intrinsic Activity EC.sub.50 (nM)
None N/A 0.64 141
[0194] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound was a partial agonist as
to MC4-R. The EC.sub.50 for MC4-R in a normal system was 238 nM and
showed intrinsic activity of 0.5.
[0195] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour decrease in food intake of 50%, and decrease in body weight of
-2%, was observed. In rat model IV feeding studies, a 24 hour
decrease in food intake of 30%, and decrease in body weight of 2%,
was observed.
[0196] In rat model IV penile erection induction experiments at 1
.mu.PK, no penile erection response was observed.
Example 15
NH.sub.2--(CH.sub.2).sub.6--C(.dbd.O)-Ser(Bzl)-D-Phe(4-Cl)-Arg-Trp-Ala-NH.-
sub.2
[0197] The peptide compound
NH.sub.2--(CH.sub.2).sub.6--C(.dbd.O)-Ser(Bzl)-D-Phe(4-Cl)-Arg-Trp-Ala-NH-
.sub.2 was synthesized by standard peptide synthesis methods as
described in International Patent Application No. PCT/US02/22196.
It was tested as described above with the results shown.
TABLE-US-00018 MC1-R MC3-R MC4-R MC5-R Inhibition at 1 .mu.M
(NDP-.alpha.-MSH) In Normal System 60% 93% 100% 79% Ki (nM)
(NDP-.alpha.-MSH) In Normal System 142 35 2 112 Low Receptor
Density System Efficacy/Intrinsic High Receptor Density System
Activity EC.sub.50 (nM) Efficacy/Intrinsic Activity EC.sub.50 (nM)
None N/A 0.67 55
[0198] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of >1 .mu.m, showed intrinsic activity of less
than 0.1, and was functionally inactive as to MC4-R.
[0199] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour decrease in food intake of 14%, and decrease in body weight of
2%, was observed. In rat model IV feeding studies, a 24 hour
decrease in food intake of 4% was observed.
[0200] In rat model IV penile erection induction experiments at 1
.mu.PK, no penile erection response was observed.
Example 16
7'-amino-heptanoyl-Ser(Bzl)-D-Phe(4-Cl)-Arg-S-(-)-1-(1-naphthyl)ethylamide
[0201] The following peptide compound was synthesized by standard
peptide synthesis methods as described in International Patent
Application No. PCT/US02/22196. It was tested as described above
with the results shown.
TABLE-US-00019 ##STR00046## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 46% 85% 98% 64% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 338 125
7 313 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A 0.63 180
[0202] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 17, showed intrinsic activity of 0.3, and was
a partial agonist as to MC4-R.
[0203] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour decrease in food intake of 13%, and decrease in body weight of
1%, was observed.
[0204] In rat model IV penile erection induction experiments at a
dose of 1 .mu.g/kg no penile erection response was observed.
Example 17
N-{3-[(S)-1-[(R)-2-Amino-3-(2,4-dichloro-phenyl)-propionyl]-4-(2-naphthale-
n-2-yl-ethyl)-piperazin-2-yl]-propyl}-guanidine
[0205] The following compound was synthesized by methods described
in U.S. patent application Ser. No. 10/762,079. It was tested as
described above with the results shown.
TABLE-US-00020 ##STR00047## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 6% 87% 99% 75% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 1198 97
3 259 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A 0.67 847
[0206] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 22, showed intrinsic activity of 0.4, and was
a partial agonist as to MC4-R.
[0207] In rat model IV feeding studies at 10 mg/kg dose levels, a
maximal 91% decrease was observed in food intake for a period of 24
hours. In rat model ICV feeding studies at 1 nmole dose level, a
maximal 32% decrease was observed in food intake for a period of 24
hours.
[0208] In mouse model IP feeding studies at 10 mg/kg dose levels, a
maximal 86% decrease was observed in food intake for a period of 4
hours.
[0209] In rat model IV penile erection induction experiments at
doses ranging from 0.3 to 30 .mu.g/kg no penile erection response
was observed. In rat model ICV penile erection induction
experiments at doses ranging from 0.3 to 3 nmole no penile erection
response was observed.
Example 18
(2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid
[(R)-2-[(S)-2-(4-amino-butyl)-4-(2-naphthalen-2-yl-ethyl)-3-oxo-piperazin-
-1-yl]-1-(2,4-dichloro-benzyl)-2-oxo-ethyl]-amide
[0210] The following compound was synthesized. It was tested as
described above with the results shown.
TABLE-US-00021 ##STR00048## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 0% 18% 92% 51% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 1370 855
50 789 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A 0.29 281
[0211] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 959, showed intrinsic activity of 0.2, and was
a partial agonist as to MC4-R.
[0212] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour decrease in food intake of 31%, and decrease in body weight of
2%, was observed.
Example 19
(2S,5R)-5-Phenyl-pyrrolidine-2-carboxylic acid
{(R)-1-(2,4-dichloro-benzyl)-2-[(2S,5R)-2-(3-guanidino-propyl)-5-(naphtha-
len-2-yloxymethyl)-3-oxo-hexahydro-pyrrolo[1,2-a]imidazol-1-yl]-2-oxo-ethy-
l}-amide
[0213] The following compound was synthesized. It was tested as
described above with the results shown.
TABLE-US-00022 ##STR00049## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 16% 43% 97% 86% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 1041 312
12 99 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A 0.22 173
[0214] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had no intrinsic
activity.
[0215] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour decrease in food intake of 38%, and decrease in body weight of
3%, was observed. In rat model IV feeding studies, a 24 hour
decrease in food intake of 1%, and decrease in body weight of 2%,
was observed.
[0216] In rat model IV penile erection induction experiments at a
dose of 1 .mu.g/kg no penile erection response was observed.
Example 20
(E)-N--[(R)-2-[(S)-2-(4-Amino-butyl)-4-(2-naphthalen-2-yl-ethyl)-3-oxo-pip-
erazin-1-yl]-1-(2,4-dichloro-benzyl)-2-oxo-ethyl]-3-phenyl-acrylamide
[0217] The following compound was synthesized. It was tested as
described above with the results shown.
TABLE-US-00023 ##STR00050## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 30% 46% 96% 60% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 502 648
6 398 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A 0.38 215
[0218] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 112, showed intrinsic activity of 0.2, and was
a partial agonist as to MC4-R.
[0219] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour decrease in food intake of 4%, and decrease in body weight of
3%, was observed.
[0220] In rat model IV penile erection induction experiments at a
dose of 1 .mu.g/kg no penile erection response was observed.
Example 21
N--[(R)-2-[(S)-2-(4-Amino-butyl)-4-(2-naphthalen-2-yl-ethyl)-3-oxo-piperaz-
in-1-yl]-1-(2,4-dichloro-benzyl)-2-oxo-ethyl]-4-phenoxy-benzamide
[0221] The following compound was synthesized. It was tested as
described above with the results shown.
TABLE-US-00024 ##STR00051## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 35% 51% 99% 51% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 915 150
1 282 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A None N/A
[0222] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had no intrinsic
activity.
[0223] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour decrease in food intake of 2%, and decrease in body weight of
1%, was observed.
[0224] In rat model IV penile erection induction experiments at a
dose of 1 .mu.g/kg no penile erection response was observed.
Example 22
(S)-1,2,3,4-Tetrahydro-isoquinoline-3-carboxylic acid
{(R)-1-(2,4-dichloro-benzyl)-2-[(2S,5R)-2-(3-guanidino-propyl)-5-(naphtha-
len-2-yloxymethyl)-3-oxo-hexahydro-pyrrolo[1,2-a]imidazol-1-yl]-2-oxo-ethy-
l}-amide
[0225] The following compound was synthesized by the methods
described in U.S. patent application Ser. No. 10/761,889. It was
tested as described above with the results shown.
TABLE-US-00025 ##STR00052## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 75% 88% 100% 96% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 111 42 1
26 Low Receptor Density System High Receptor Density System
Efficacy/Intrinsic Efficacy/Intrinsic Activity EC.sub.50 (nM)
Activity EC.sub.50 (nM) None N/A 0.30 64
[0226] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system a
pA.sub.2 value of 8.1, and was an antagonist as to MC4-R.
[0227] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour decrease in food intake of 22%, was observed.
[0228] In rat model IV penile erection induction experiments at 1
.mu.PK, no penile erection response was observed.
Example 23
Heptanoyl-Thr(Bzl)-D-Phe(4-Cl)-Arg-Trp-NH.sub.2
[0229] The peptide compound
Heptanoyl-Thr(Bzl)-D-Phe(4-Cl)-Arg-Trp-NH.sub.2 was synthesized by
standard peptide synthesis methods as described in International
Patent Application No. PCT/US02/22196. It was tested as described
above with the results shown.
TABLE-US-00026 MC1-R MC3-R MC4-R MC5-R Inhibition at 1 .mu.M
(NDP-.alpha.-MSH) In Normal System 28% 39% 91% 65% Ki (nM)
(NDP-.alpha.-MSH) In Normal System 747 374 21 292 Low Receptor
Density System Efficacy/Intrinsic High Receptor Density System
Activity EC.sub.50 (nM) Efficacy/Intrinsic Activity EC.sub.50 (nM)
None N/A 0.75 184
[0230] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of >1000, and was a partial agonist as to
MC4-R with an intrinsic activity of 0.4.
[0231] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour decrease in food intake of 18%, and decrease in body weight of
1%, was observed.
[0232] In rat model IV penile erection induction experiments at a
dose of 1 .mu.g/kg no penile erection response was observed.
Example 24
2-naphthylacetyl-1-amino-1-cyclohexane-carbonyl-D-Phe(4-Cl)-Arg-Trp-NH.sub-
.2
[0233] The peptide compound
2-naphthylacetyl-1-amino-1-cyclohexane-carbonyl-D-Phe(4-Cl)-Arg-Trp-NH.su-
b.2 was synthesized by standard peptide synthesis methods as
described in International Patent Application No. PCT/US02/22196.
It was tested as described above with the results shown.
TABLE-US-00027 MC1-R MC3-R MC4-R MC5-R Inhibition at 1 .mu.M
(NDP-.alpha.-MSH) In Normal System 25% 35% 92% 63% Ki (nM)
(NDP-.alpha.-MSH) In Normal System 897 453 40 300 Low Receptor
Density System Efficacy/Intrinsic High Receptor Density System
Activity EC.sub.50 (nM) Efficacy/Intrinsic Activity EC.sub.50 (nM)
None N/A None N/A
[0234] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of >1000, and was a partial agonist as to
MC4-R with an intrinsic activity of 0.2.
[0235] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour decrease in food intake of 21%, and decrease in body weight of
1%, was observed.
Example 25
N-{1(R)-(2,4-Dimethyl-benzyl)-2-[2(S)-(3-guanidino-propyl)-5(R)-methyl-4-(-
2-naphthalen-2-yl-ethyl)-piperazin-1-yl]-2-oxo-ethyl}-acetamide
[0236] The following compound was synthesized by the method of
Scheme 5 of U.S. patent application Ser. No. 10/837,519, using
2-naphthylacetic acid as J-COOH, D-alaninol as
NH.sub.2--CH(R.sub.5)--CH(R.sub.4)--OH, D-alanine methyl ester as
NH.sub.2--CH(R.sub.5--COOCH.sub.3, and Boc-D-2,4-dimethyl-Phe-OH as
Q-COOH. An acetyl group was attached to the amino group of
D-2,4-dimethyl-Phe residue by the method described in Example 36 of
U.S. patent application Ser. No. 10/837,519. It was tested as
described above with the results shown. The mass was analyzed as
571.9 (M+H).
TABLE-US-00028 ##STR00053## Ki (nM) MC1-R MC3-R MC4-R MC5-R 568 74
1 43 Inhibition at 1 .mu.M (NDP-.alpha.-MSH) In Normal System MC1-R
MC3-R MC4-R MC5-R 28% 75% 100% 89% Low Receptor Density System High
Receptor Density System Efficacy/Intrinsic Efficacy/Intrinsic
Activity EC.sub.50 (nM) Activity EC.sub.50 (nM) None N/A None
N/A
[0237] In a cAMP assay using MC4-R, at 1 .mu.M concentrations the
compound of Example 26 exhibited no intrinsic activity. It tested
as an MC4-R antagonist in a normal system with a pA.sub.2 value of
8.3.
[0238] In mouse model IP feeding studies at 10 mg/kg dose levels, a
4 hour decrease in food intake of 34% was observed.
Example 26
N-{3-[1-[2(R)-Amino-3-(2-methyl,
4-chloro-phenyl)-propionyl]-5(R)-methyl-4-(2-naphthalen-2-yl-ethyl)-piper-
azin-2(S)-yl]-propyl}-guanidine
[0239] The following compound was synthesized by the methods of
U.S. patent application Ser. No. 10/837,519 using 2-naphthylacetic
acid as J-COOH, (R)-(-)-2-amino-1-propanol as
NH.sub.2--CH(R.sub.5)--CH(R.sub.4)--OH, Fmoc-Arg(Boc).sub.2-OH as
Prt-NH--C(R.sub.2)--COOH, D-alanine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3 and Boc-D-2-methyl,
4-chloro-Phe-OH as Q-COOH. It was tested as described above with
the results shown. The mass was analyzed as 549 (M+H).
TABLE-US-00029 ##STR00054## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 6% 74% 100% 77% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 1052 99
1 219 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) None N/A High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) 0.78 31
[0240] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 214, showed intrinsic activity of 0.3, and was
a partial agonist as to MC4-R.
[0241] In mouse model IP feeding studies at 3 and 10 mg/kg dose
levels, a maximum 44% and 86% decrease, respectively, was observed
in food intake for a period of 4 hours.
[0242] In rat model IV penile erection induction experiments at 1
and 3 mg/kg, no penile erection were observed.
Example 27
N-{3-[1-[2(R)-Amino-3-(2,4-dimethyl-phenyl)-propionyl]-5(R)-methyl-4-(2-na-
phthalen-2-yl-ethyl)-piperazin-2(S)-yl]-propyl}-guanidine
[0243] The following compound was synthesized by the methods of
U.S. patent application Ser. No. 10/837,519 using 2-naphthylacetic
acid as J-COOH, (R)-(-)-2-amino-1-propanol as
NH.sub.2--CH(R.sub.5)--CH(R.sub.4)--OH, Fmoc-Arg(Boc).sub.2-OH as
Prt-NH--C(R.sub.2)--COOH, D-alanine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3 and Boc-D-2,4-dimethyl-Phe-OH as
Q-COOH. It was tested as described above with the results shown.
The mass was analyzed as 529 (M+H).
TABLE-US-00030 ##STR00055## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 9% 62% 100% 59% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 1233 167
11 665 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) None N/A High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) 0.61 129
[0244] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 321, showed intrinsic activity of 0.2, and was
a partial agonist as to MC4-R.
[0245] In mouse model IP feeding studies at 3 and 10 mg/kg dose
levels, a maximum 47% and 88% decrease, respectively, was observed
in food intake for a period of 4 hours.
[0246] In rat model IV penile erection induction experiments at 1
and 3 mg/kg, no penile erections were observed. In rat model ICV
penile erection induction experiments at doses of 1 and 3 nmole no
penile erection response was observed.
Example 28
N-{3-[1-[2(R)-Amino-3-(4-chloro-phenyl)-propionyl]-5(R)-methyl-4-(2-naphth-
alen-2-yl-ethyl)-piperazin-2(S)-yl]-propyl}-guanidine
[0247] The following compound was synthesized by the methods of
U.S. patent application Ser. No. 10/837,519 using 2-naphthylacetic
acid as J-COOH, (R)-(-)-2-amino-1-propanol as
NH.sub.2--CH(R.sub.5)--CH(R.sub.4)--OH, Fmoc-Lrg(Boc).sub.2-OH as
Prt-NH--C(R.sub.2)--COOH, D-alanine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3 and Boc-D-4-chloro-Phe-OH as
Q-COOH. It was tested as described above with the results shown.
The mass was analyzed as 535 (M+H).
TABLE-US-00031 ##STR00056## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 64% 70% 97% 61% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 94 160
11 551 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) 0.29 106 High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) 0.75 24
[0248] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 252, showed intrinsic activity of 0.8, and was
an agonist as to MC4-R.
[0249] In mouse model IP feeding studies at 3 mg/kg dose levels, a
maximum 46% decrease was observed in food intake for a period of 4
hours.
[0250] In rat model IV penile erection induction experiments at
doses ranging 0.003 to 3 mg/kg, no penile erections were
observed.
Example 29
N-{3-[1-[2(R)-Amino-3-(4-chloro-phenyl)-propionyl]-5(R)-methyl-4-(2-naphth-
alen-2-yl-ethyl)-piperazin-2(S)-yl]-propyl}-guanidine
[0251] The following compound was synthesized by the method of
Scheme 7 of U.S. patent application Ser. No. 10/837,519 using
2-naphthyl acetic acid as J-COOH, L-Orn(Boc) methyl ester as
NH.sub.2--CH(R.sub.2)--COOCH.sub.3, and Boc-D-2,4-di-methyl-Phe-OH
as Q-COOH. It was tested as described above with the results shown.
The mass was analyzed as 543 (M+H).
TABLE-US-00032 ##STR00057## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 0% 25% 85% 7% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 431 526
6 1536 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) 0.25 45 High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) 0.77 34
[0252] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 46, showed intrinsic activity of 0.8, and was
an agonist as to MC4-R.
[0253] In mouse model IP feeding studies at 3 and 10 mg/kg dose
levels, a 3% and 26% decrease, respectively, was observed in food
intake for a period of 20 hours.
[0254] In rat model IV penile erection induction experiments at
doses ranging 0.0003 to 0.03 mg/kg, it exhibited a maximum of 0.5
mean PEs per rat with no more than 50% animals responding.
Example 30
N-{(R)-1-(2,4
dimethyl-benzyl)-2-[(2S,5R)-2-(3-guanidino-propyl)-5-methyl-4-(2-naphthal-
en-2-yl-acetyl)-piperazin-1-yl]-2-oxo-ethyl}-acetamide
[0255] The following compound was synthesized by the method used
for Example 29, with an acetyl group introduced at the amino group
of D-2,4-dimethyl-Phe by reaction of the compound of Example 29
with Ac-OSu in DMF. It was tested as described above with the
results shown. The mass was analyzed as 585 (M+H).
TABLE-US-00033 ##STR00058## Ki (nM) (NDP-.alpha.-MSH) In Normal
System MC1-R MC3-R MC4-R MC5-R 1883 232 11 250 Low Receptor Density
System Efficacy/Intrinsic Activity EC.sub.50 (nM) None N/A High
Receptor Density System Efficacy/Intrinsic Activity EC.sub.50 (nM)
0.18 51
[0256] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 9, showed intrinsic activity of 0.4, and was a
partial agonist as to MC4-R.
[0257] In mouse model IP feeding studies at 3 mg/kg dose levels, a
maximum 8% decrease was observed in food intake for a period of 4
hours.
[0258] In rat model IV penile erection induction experiments at
doses ranging 0.0003 to 0.03 mg/kg, it exhibited no penile activity
response.
Example 31
N-{(R)-1-(2-methyl,
4-chloro-benzyl)-2-[(2S,5R)-2-(3-guanidino-propyl)-5-methyl-4-(2-naphthal-
en-2-yl-acetyl)-piperazin-1-yl]-2-oxo-ethyl}-acetamide
[0259] The following compound was synthesized by the method of
Scheme 7 of U.S. patent application Ser. No. 10/837,519 using
2-naphthyl acetic acid as J-COOH, L-Orn(Boc) methyl ester as
NH.sub.2--CH(R.sub.2)--COOCH.sub.3, and
Boc-D-4-chloro-2-methyl-Phe-OH as Q-COOH with an acetyl group
introduced at the amino group of the D-4-chloro-2-methyl-Phe
residue by reaction of the compound with Ac-OSu in DMF. It was
tested as described above with the results shown. The mass was
analyzed as 605 (M+H).
TABLE-US-00034 ##STR00059## Ki (nM) (NDP-.alpha.-MSH) In Normal
System MC1-R MC3-R MC4-R MC5-R 1388 136 5 234 Low Receptor Density
System Efficacy/Intrinsic Activity EC.sub.50 (nM) None N/A High
Receptor Density System Efficacy/Intrinsic Activity EC.sub.50 (nM)
0.27 39
[0260] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 3, showed intrinsic activity of 0.6, and was a
partial agonist as to MC4-R.
[0261] In mouse model IP feeding studies at 10 mg/kg dose levels, a
maximum 7% decrease, was observed in food intake for a period of 4
hours.
[0262] In rat model IV penile erection induction experiments at
doses ranging 0.0003 to 0.03 mg/kg, it exhibited no penile activity
response.
Example 32
N-{(R)-2-[(2S,5R)-2-(3-guanidino-propyl)-5-methyl-4-(2-naphthalen-2-yl-ace-
tyl)-piperazin-1-yl]-1-naphthalen-2-ylmethyl-2-oxo-ethyl}-acetamide
[0263] The following compound was synthesized by the method of
Scheme 6 of U.S. patent application Ser. No. 10/837,519 using
2-naphthylacetic acid as J-COOH, D-alanine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3, and Boc-D-2-Nal-OH as Q-COOH
with an acetyl group attached to the amino group of D-2-Nal by
reaction of the compound with Ac-OSu in DMF. It was tested as
described above with the results shown. The mass was analyzed as
605 (M+H).
TABLE-US-00035 ##STR00060## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 51% 38% 97% 44% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 406 634
7 826 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) None N/A High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) None N/A
[0264] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 7, showed intrinsic activity of 0.3, and was a
partial agonist as to MC4-R.
[0265] In mouse model IP feeding studies at 10 mg/kg dose levels, a
maximum 34% decrease was observed in food intake for a period of 4
hours.
[0266] In rat model ICV penile erection induction experiments at
doses of 0.1 and 1 nmole, it exhibited no penile erection
response.
Example 33
N-{3-[(2S)-1-[(R)-2-Amino-3-(naphthlene-2-yl)-propionyl]-5(R)-methyl-4-(2--
naphthalen-2-yl-acetyl)-piperazin-2-yl]-propyl}-guanidine
[0267] The following compound was synthesized by the method of
Scheme 6 of U.S. patent application Ser. No. 10/837,519 using
2-naphthylacetic acid as J-COOH, D-alanine methyl ester as
NH.sub.2--CH(R.sub.5)--COOCH.sub.3, and Boc-D-2-Nal-OH as Q-COOH.
It was tested as described above with the results shown. The mass
was analyzed as 605 (M+H).
TABLE-US-00036 ##STR00061## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 43% 40% 89% 50% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 227 596
19 548 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) None N/A High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) 0.73 50
[0268] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 42, showed intrinsic activity of 1, and was an
agonist as to MC4-R.
[0269] In mouse model IP feeding studies at 10 mg/kg dose levels, a
maximum 25% decrease was observed in food intake for a period of 4
hours.
[0270] In rat model IV penile erection induction experiments at
doses of 0.0003 to 0.03 mg/kg, it exhibited no penile erection
response.
Example 34
Ac-Nle-cyclo(Asp-His-D-Nal 2-Arg-Nal 2-Lys)-OH
[0271] The following peptide compound was synthesized by standard
peptide synthesis methods. It was tested as described above with
the results shown.
TABLE-US-00037 ##STR00062## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 102% 98% 100% 100% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 0.2 0.3
0.02 0.2 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) None N/A High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) None N/A
[0272] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound was inactive at 1 nM
dose. It tested as an MC4-R antagonist in a normal system with a
pA.sub.2 value of 8.1. In separate experiments, human MC1-R was
used to determine the Ki (nM) (NDP-.alpha.-MSH) in a normal system,
with a Ki of 0.25 nM.
[0273] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour increase in food intake of 12 g, and increase in body weight
of 16 g, was observed.
[0274] In an ICV rat model of PE efficacy the compound was inactive
when administered at a dose of 3 nanomole.
Example 35
Ac-cyclo(Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH--CH.sub.3
[0275] The following peptide compound was synthesized by standard
peptide synthesis methods. It was tested as described above with
the results shown.
TABLE-US-00038 ##STR00063## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 67% 99% 100% 98% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 643 3
0.06 6 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) None N/A High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) None N/A
[0276] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound was inactive at 1 nM
dose.
[0277] In rat model ICV feeding studies at 1 nmol dose levels, a 24
hour increase in food intake of 6 g, and increase in body weight of
6 g, was observed. In mouse model IP feeding studies at 1 mg/kg
dose levels, a maximal 4% decrease was observed in food intake for
a period of 4 hours.
[0278] In an ICV rat model of PE efficacy, the compound was
inactive when administered at a dose of 3 nM.
Example 36
Ac-Nle-cyclo(-Asp-Trp-D-Phe-Arg-Nal 1-Lys)-N(CH.sub.3).sub.2
[0279] The following peptide compound was synthesized by standard
peptide synthesis methods. It was tested as described above with
the results shown.
TABLE-US-00039 ##STR00064## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 71% 83% 99% 92% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 12 231
38 31 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) None N/A High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) 0.98 4
[0280] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 272, showed intrinsic activity of 0.8, and was
an agonist as to MC4-R. In separate experiments, human MC1-R was
used to determine the Ki (nM) (NDP-.alpha.-MSH) in a normal system,
with a Ki of 39 nM.
Example 37
Ac-Nle-cyclo(-Asp-His-D-Phe-Arg-Nal 1-Lys)-N(CH.sub.3).sub.2
[0281] The following peptide compound was synthesized by standard
peptide synthesis methods. It was tested as described above with
the results shown.
TABLE-US-00040 ##STR00065## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 82% 92% 100% 92% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 0.39 111
5 50 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) 0.69 2.1 High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) 0.88 0.6
[0282] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 44, showed intrinsic activity of 0.7, and was
an agonist as to MC4-R. In separate experiments, human MC1-R was
used to determine the Ki (nM) (NDP-.alpha.-MSH) in a normal system,
with a Ki of 0.08 nM.
Example 38
Ac-Nle-cyclo(-Asp-Trp-D-Phe-Arg-Nal
1-Lys)-NH--CH.sub.2--CH.sub.3
[0283] The following peptide compound was synthesized by standard
peptide synthesis methods. It was tested as described above with
the results shown.
TABLE-US-00041 ##STR00066## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 98% 92% 100% 92% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 4.2 77 9
47 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) None N/A High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) 1.0 10.0
[0284] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 120, showed intrinsic activity of 0.6, and was
an agonist as to MC4-R. In separate experiments, human MC1-R was
used to determine the Ki (nM) (NDP-.alpha.-MSH) in a normal system,
with a Ki of 30 nM.
Example 39
Ac-Nle-cyclo(-Asp-His-D-Phe-Arg-Nal
1-Lys)-NH--CH.sub.2--CH.sub.3
[0285] The following peptide compound was synthesized by standard
peptide synthesis methods. It was tested as described above with
the results shown.
TABLE-US-00042 ##STR00067## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 100% 93% 98% 93% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 0.07 28
2 63 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) 0.77 10 High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) 1.0 1.1
[0286] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 0.2, showed intrinsic activity of 0.6, and was
a partial agonist as to MC4-R. In separate experiments, human MC1-R
was used to determine the Ki (nM) (NDP-.alpha.-MSH) in a normal
system, with a Ki of 0.04 nM.
Example 40
Ac-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-NH--CH.sub.2--CH.sub.3
[0287] The following peptide compound was synthesized by standard
peptide synthesis methods. It was tested as described above with
the results shown.
TABLE-US-00043 ##STR00068## Inhibition at 1 .mu.M (NDP-.alpha.-MSH)
In Normal System MC1-R MC3-R MC4-R MC5-R 96% 65% 96% 16% Ki (nM)
(NDP-.alpha.-MSH) In Normal System MC1-R MC3-R MC4-R MC5-R 5 84 6
>1,000 Low Receptor Density System Efficacy/Intrinsic Activity
EC.sub.50 (nM) 1.0 3.1 High Receptor Density System
Efficacy/Intrinsic Activity EC.sub.50 (nM) 1.0 0.5
[0288] In a cAMP assay for determination of functionality as to
MC4-R, it was determined that the compound had in a normal system
an EC.sub.50 (nM) of 3, showed intrinsic activity of 0.6, and was a
partial agonist as to MC4-R. In separate experiments, human MC1-R
was used to determine the Ki (nM) (NDP-.alpha.-MSH) in a normal
system, with a Ki of 0.52 nM.
[0289] In rat model IV penile erection induction experiments, the
compound was active and showed peak PE activity when administered
at a dose of 0.3 mg/kg, with 2.5 mean penile erections per rat
observed with 83% of the animals responding.
[0290] The preceding examples can be repeated with similar success
by substituting the generically or specifically described reagents,
reactions and/or operating conditions of this invention for those
used in the preceding examples. In particular, and without limiting
the generality of the foregoing, it is possible to use other
methods of transformation, transfection or expressing differential
densities or levels of melanocortin receptors, other sources of
melanocortin receptors, or cell types and systems, other assay
systems, other methods of determining functional activity, and the
like.
[0291] Although the invention has been described in detail with
particular reference to these preferred embodiments, other
embodiments can achieve the same results. Variations and
modifications of the present invention will be obvious to those
skilled in the art and it is intended to cover in the appended
claims all such modifications and equivalents. The entire
disclosures of all references, applications, patents, and
publications cited above are hereby incorporated by reference as if
set forth in full.
Sequence CWU 1
1
2113PRTHomo sapiensMOD_RES(1)..(1)Acetylation 1Ser Tyr Ser Met Glu
His Phe Arg Trp Gly Lys Pro Val1 5 10219DNAArtificialTetracycline
operator 2 2tccctatcag tgatagaga 19
* * * * *